Novel plant genes

ABSTRACT

The present invention relates to a DNA which encodes a polypeptide having flavonoid-3′,5′-hydroxylase activity, a recombinant DNA containing said DNA, and a plant having a pigment pattern which the plant does not originally have and which is acquired by transformation with said recombinant DNA.

TECHNICAL FIELD

[0001] The present invention relates to a technique to breed plants orplant cells using recombinant DNA technology. More particularly, thepresent invention relates to a technique to breed novel plant cells ornovel plants which show exogenous pigment patterns by transforming plantcells or plants with a recombinant DNA containing a DNA which encodes apolypeptide having flavonoid-3′,5′-hydroxylase activity (hereinafteralso referred to as the DNA encoding flavonoid-3′,5′-hydroxylase).

BACKGROUND ART

[0002] Crossing between varieties has been conventionally employed as amethod for altering the color of flowers and fruits of plants. However,crossing is carried out between varieties of the same genus, and usuallyof the same species, and therefore, it is extremely difficult to givespecific colors to certain plant species. For example, in spite oflongtime efforts of breeders, no one has yet successfully bred a bluerose or a blue carnation.

[0003] In recent years, recombinant DNA technology has enabled plantbreeding between different species or genus, and it is expected to breednew plants having unprecedented pigment patterns which can not beobtained by the conventional breeding methods by crossing (PlantMolecular Biology, vol.13, p.287-294, 1989). For example, it is reportedthat petunia showing unprecedented brick-red color on flowers was bredby cloning a gene encoding dihydroflavonol-4-reductase, which is anenzyme participating in pigment biosynthetic pathway, from maize andintroducing it into petunia (Japanese Published Unexamined PatentApplication No. 2305/90; Nature, vol.330, p.677-678, 1987). Further, areport has been made of a case in which new pigment patterns wereproduced by introducing the chalcone synthase gene of petunia at thesense or anti-sense orientation to partially inhibit the expression ofthe gene (Nature, vol.333, p.866-869, 1988; The Plant Cell, vol.2,p.279-289, 1990; The Plant Cell, vol.2, p.291-299, 1990).

[0004] Biosynthetic pathways for anthocyanins, which contribute to blueor red color of flowers, have been studied genetically and biochemicallyin detail using petunia and others (Petunia, Edited by K. C. Sink,Springer Verlag, p.49-76, 1984; The Flavonoids, Edited by J. B.Harborne, Chapman and Hall, p.399-425, 1988; Molecular Approaches toCrop Improvement, Edited by E. S. Denis and D. J. Rewerin, SpringerVerlag, p.127-148, 1991). As a result of these studies, it is shown thatthe presence/absence of hydroxyl group at the 3′- and 5′-positions ofthe B ring of anthocyanin greatly affects the color of flowers, and alsoit is shown that, generally, the blue color of flowers is intensified asthe B ring is hydroxylated in a higher degree. The hydroxylation of theB ring of anthocyanins occurs at the stage of their precursors,flavanones or dihydroflavonols. As enzymes which catalyze thishydroxylation, two types of enzyme have been known;flavonoid-3′-hydroxylase which hydroxylates only the 3′-position of theB ring, and flavonoid-3′,5′-hydroxylase which hydroxylates both the 3′-and 5′-positions. Petunia with blue flowers has both the enzymes, butthat with red flowers has only the former one. Plants like roses,carnations, and chrysanthemums do not have anthocyanins which have Bring hydroxylated at both the 3′- and 5′-positions, and therefore areconsidered not to have the latter type of enzyme.

[0005] These hydroxylases are localized in the microsomal membrane andrequire NADPH as a coenzyme. They are presumed to be members of thecytochrome P450 enzyme group on the basis of their behavior againstvarious inhibitors (The Flavonoids, Edited by J. B. Harborne, Chapmanand Hall, p.399-425, 1988; Molecular Approaches to Crop Improvement,Edited by E. S. Denis and D. J. Rewerin, Springer Verlag, p.127-148,1991).

[0006] Cytochrome P450 is an enzyme group which is widely distributedamong eucaryotes and procaryotes and which is involved in thebiosynthesis of important lipids such as steroids and in the oxidativemetabolism of lipophilic substances. In higher animals, it forms a superfamily consisting of one hundred or more molecular species (J. Biol.Chem., vol.266, p.13469-13472, 1991; Pharmacol. Rev., vol.40, p.243-288,1988). In plants, cinnamic acid-4-hydroxylase and kaurene oxidase areconsidered to belong to the cytochrome P450 group (Plant Physiol.,vol.96, p.669-674, 1991). Further, a gene encoding a cytochrome P450enzyme whose function is unknown has been cloned from avocado (Proc.Natl. Acad. Sci. USA, vol.87, p.3904-3908, 1990). As a result of thecomparison of the amino acid sequences of various types of cytochromeP450 enzymes, it is known that the sequence of the heme-binding site isconserved (Proc. Natl. Acad. Sci. USA, vol.85, p.7221-7225, 1988;Pharmacol. Rev. vol.40, p.243-288, 1988).

[0007] In petunia, flavonoid-3′,5′-hydroxylase is encoded by twodominant genes called Hf-1 and Hf-2. The enzymes encoded by the genesare isozymes, and the degree of expression of Hf-1 is higher (Petunia,Edited by K. C. Sink, Springer Verlag, p.49-76, 1984). Further,characteristics of said enzyme of Verbena have been reported (Z.Naturforschung, vol.37c, p.19-23, 1982).

[0008] It is also reported that 3′,5′-hydroxylase, a key enzyme in thebiosynthesis of delphinidin, which is a blue pigment in petunia, hasbeen successfully cloned (Nikkei Biotech, Aug. 26, 1991). However, noreport has been made yet of a case in which the cloned gene of saidenzyme is allowed to express in a plant to alter pigments in the plant.

DISCLOSURE OF THE INVENTION

[0009] The present invention provides a DNA which encodes a polypeptidehaving flavonoid-3′,5′-hydroxylase activity which is represented by theamino acid sequence shown by SEQ ID NO: 1, 63 or 64, a DNA whichhybridizes with said DNA, a recombinant DNA constructed by incorporatingany of these DNAs or a part of their sequences into a vector DNA, andplant cells or plants which carry said recombinant DNA.

[0010] It is possible to breed plants having novel pigment patterns byintroducing said DNA, i.e., a DNA that encodes a polypeptide havingflavonoid-3′,5′-hydroxylase activity, into plant cells or plants by theuse of recombinant DNA technology.

[0011] The DNA of the present invention may be any DNA which encodes apolypeptide having flavonoid-3′,5′-hydroxylase activity, i.e., a DNAwhich encodes a polypeptide represented by the amino acid sequence shownby SEQ ID NO: 1, 63 or 64, or a DNA which hybridizes with said DNA(hereinafter referred to as hDNA). The hDNA may be any DNA whichhybridizes with the DNA encoding the polypeptide represented by theamino acid sequence shown by SEQ ID NO: 1, 63 or 64 in 2×SSC (0.3 Msodium chloride, 0.03 M sodium citrate, pH 7.0) at 50° C.

[0012] The DNA which encodes the polypeptide represented by the aminoacid sequence shown by SEQ ID NO: 63 or 64 hybridizes with the DNA whichencodes the polypeptide represented by the amino acid sequence shown bySEQ ID NO: 1 under the above-mentioned conditions.

[0013] Other examples of the DNAs of the present invention include DNAswherein a part of the nucleotide sequence of the above-mentioned DNAs isdeleted or replaced with other nucleotide sequences, as far as such DNAsencode a polypeptide having flavonoid-3′,5′-hydroxylase activity.

[0014] Examples of the DNA sources include a genomic DNA of plants whichhave flavonoid-3′,5′-hydroxylase, and a cDNA which is synthesized froman mRNA extracted from the expression sites of said enzyme using areverse transcriptase. Examples of the plants having said enzyme includepetunia (Solanaceae), pansy (Violaceae), primrose (Primulaceae),delphinium (Ranunculaceae), sweet pea (Leguminosae), Japanese gentian(Gentianaceae), balloon flower (Campanulaceae), forget-me-not(Boraginaceae), hydrangea (Saxifragaceae), verbena (Verbenaceae),dayflower (Commelinaceae), iris (Iridaceae), hyacinth (Liliaceae),Russell prairie gentian (Gentianaceae), and campanula (Campanulaceae).

[0015] In the present invention, on the basis of the presumption thatflavonoid-3′,5′-hydroxylase is a member of the cytochrome P450 family,DNA sequences encoding the amino acid sequence of the heme-binding siteof cytochrome P450 (hereinafter referred to as the core sequence) areamplified and isolated using the PCR method.

[0016] The core sequence is the region that shows high homology amongdifferent molecular species of cytochrome P450 and among those ofdifferent organisms. More than 80% of the sequences for cytochrome P450which have been ever isolated have the core sequence shown in FIG. 1(DNASIS™ Data Base CD, 009-1 and 2, Hitachi Software Engineering Co.,Ltd., 1990). DNA sequences which can encode the amino acid sequence ofthe region indicated by arrows are hypothesized. Then, in order toamplify and isolate the DNA sequences encoding this region by the PCRmethod, 16 types of sense primers shown by SEQ ID NO: 2 to 17, and 12types of antisense primers shown by SEQ ID NO: 18 to 29 are chemicallysynthesized. The sense primers are synthetic DNA primers each consistingof 18 bases, and each sense primer has, at the 3′ end side, one of the16 types of 8-base DNA sequences at the 3′ end encoding Pro-Phe-Gly orPro-Phe-Ser, and has, at the 5′ end side, a 10-base DNA sequence whichincludes a recognition site for a restriction enzyme, EcoRI. Theantisense primers are synthetic DNA primers each consisting of 18 bases,and each primer has, at the 3′ end side, one of the 12 types ofsequences which are inversely linked to the 8-base DNA sequence at the3′ end encoding Cys-Xxx-Gly (wherein Xxx represents Ile, Leu, Val, Ala,or Pro), and has, at the 5′ end side, a 10-base DNA sequence including arecognition site for a restriction enzyme, BamHI.

[0017] By the use of PCR in which these synthetic DNA primers areemployed in combination, various DNA fragments which encode the coresequence can be amplified and isolated, and their DNA sequences can bedetermined. As cytochrome P450 forms a super family which consists ofvarious molecular species, it is expected that various types of coresequences can be obtained from one template DNA. During the process ofthe present invention, 15 types of core sequences shown by SEQ ID NO: 30to 44 were obtained.

[0018] It is necessary to make a presumption as to which core sequenceis the target sequence among the thus obtained core sequences. In thepresent invention, the target sequence is presumed by investigatingwhether the expression/non-expression of each core sequence isgenetically linked to the presence/absence of said enzyme activity. Inorder to investigate the genetic linkage, a petunia which originally hassaid enzyme (a blue flower cultivar) is backcrossed with a mutantpetunia variety which lacks said enzyme (a red flower cultivar) toproduce a genetically segregating population (1:1) regarding thepresence/absence of said enzyme. Then, the mode of expression of eachcore sequence in the petals of individual plants in this population isinvestigated. If the mode of expression of any core sequence agrees with(is genetically linked with) the presence/absence of said enzyme, thecore sequence is presumed to be a part of the gene encoding said enzyme.

[0019] In order to investigate whether a core sequence is expressed inthe petals, the present invention uses a method called SSP (singlespecific primer) polymerase chain reaction (PCR). SSP.PCR is a methoddescribed in Biochemistry Biophysics Research Communication, vol.167,p.504-506, 1990. By the use of this method, it is possible to amplify aDNA sequence flanking a core sequence and to determine thepresence/absence of the corresponding product. First, specific DNAprimers are synthesized based on the DNA sequences encoding the coresequences. In the present invention, 15 types of DNAs (K primers 01 to15) shown by SEQ ID NO: 45 to 59 were synthesized and used as thespecific DNA primers. Then, cDNAs are prepared from the petals of eachpetunia plant in the backcrossed population, digested with appropriaterestriction enzymes, and then ligated with appropriate double-strandsynthetic DNA (called cassette) which had the corresponding cleaved endsusing a ligase to prepare templates. In the present invention, syntheticDNAs shown by SEQ ID NO: 60 and 61 were annealed and used as thecassette. The synthetic DNA shown by SEQ ID NO: 60 was also used as theprimer for the cassette. With the template DNA ligated to the cassette,PCR was carried out between the specific primer and the primer for thecassette, whereby the DNA sequence flanking the core sequence isamplified. The presence/absence of its product reflects theexpression/non-expression of the core sequence.

[0020] As a result of the search in the petunia population obtained bythe backcrossing, it was revealed that the presence/absence of a product(approximately 85 bp) which was amplified by SSP.PCR using the specificprimer (K14) shown by SEQ ID NO: 58 was completely linked with thepresence/absence of said enzyme activity. As this primer was designedbased on the core sequence shown by SEQ ID NO: 43, this sequence isassumed to be the core sequence of said enzyme. On the basis of SEQ IDNO: 43, the primer (J14) shown by SEQ ID NO: 62 was synthesized andSSP.PCR was carried out. As a result, the presence/absence of a productof approximately 280 bp was completely linked with the presence/absenceof the enzyme activity. This result strongly suggests that the coresequence shown by SEQ ID NO: 43 is the target sequence.

[0021] The product of approximately 280 bp thus amplified is assumed tobe a part of the cDNA sequence that encodes said enzyme. The full lengthcDNA sequence shown by SEQ ID NO: 1 can be obtained by preparing petuniaflower cDNA library according to the method described in a book byManiatis et al., and then searching the library using theabove-mentioned product as a probe. If the expression of the obtainedsequence in a plant which originally does not have said enzyme resultsin the detection of said enzyme activity in the plant, it will be provedthat this sequence is the DNA sequence encoding the polypeptide havingsaid enzyme activity. In the present invention, the DNA shown by SEQ IDNO: 1 was introduced into tobacco and petunia cultivars both of which donot have said enzyme, and expressed. As a result, said enzyme activitywas detected in both plants, and thus the DNA was proved to be the DNAencoding the polypeptide having said enzyme activity.

[0022] Cloning of DNAs can be carried out using a material such as acDNA which is synthesized based on an mRNA extracted from the petals ofpetunia using a reverse transcriptase.

[0023] DNA cloning and DNA analysis can be carried out according togeneral techniques described in Molecular Cloning a Laboratory ManualSecond Edition, J. Sambrook, E. F. Frisch, T. Maniatis, Cold SpringHarbor Laboratory Press, 1989 (hereinafter referred to as the book byManiatis et al.), and the like.

[0024] PCR can be carried out according to ordinary techniques describedin PCR Technology, Edited by H. A. Ehrlich, Stockton Press, 1989, PCRProtocols, Edited by M. A. Innis, D. H. Gerfand, J. J. Sninsky, and T.J. White, Academic Press, 1990, and the like.

[0025] Determination of nucleotide sequences can be carried outaccording to methods using the Taq Dideoxy™ Terminator Cycle SequencingKit (ABI Co., Ltd.) and the Model 373A DNA Sequencing System (ABI Co.,Ltd.), and the like.

[0026] DNA fragments encoding polypeptides which have analogoussequences and said enzyme activity can be cloned from any of the plantsmentioned above as the DNA source by an ordinary method using, as aprobe for hybridization, the whole or a part of the DNA sequence shownby SEQ ID NO: 1 which encodes the polypeptide having said enzymeactivity and is derived from petunia as above.

[0027] In the present invention, according to the above-mentionedmethod, a DNA which encodes a polypeptide having the amino acid sequenceshown by SEQ ID NO: 63 has been cloned from Russell prairie gentian, anda DNA which encodes a polypeptide having the amino acid sequence shownby SEQ ID NO: 64 has been cloned from campanula.

[0028] New coloration can be introduced into a host plant which does nothave said enzyme by introducing a DNA fragment which encodes apolypeptide having said enzyme activity into the host plant, allowing itto express, and thereby hydroxylating the 3′- and 5′-positions ofanthocyanin pigments. Examples of such host plants include rose(Rosaceae), carnation (Caryophyllaceae), petunia (Solanaceae), tobacco(Solanaceae), chrysanthemum (Compositae), stock (Cruciferae), begonia(Begoniaceae), snapdragon (Scrophulariaceae), camellia (Theaceae), lily(Liliaceae), and orchid (Orchidaceae).

[0029] Further, in plant species which originally have said enzyme, theenzyme activity can be inhibited by introducing said DNA fragment at theantisense or sense orientation and allowing it to express (Nature,vol.333, p.866-869, 1988; The Plant Cell, vol.2, p.279-289, 1990; ThePlant Cell, vol.2, p.291-299, 1990). By application of such methods,breeding of a plant species having an unprecedented pigment pattern canbe achieved.

[0030] In order to introduce the DNA fragment which encodes thepolypeptide having said enzyme activity into plants and allow it toexpress, it is necessary to introduce an appropriate promoter at thesite upstream of the region encoding the polypeptide having said enzymeactivity. An example of a promoter that works in plants is 35 S promoterof Cauliflower Mosaic Virus (CaMV) (Cell, vol.21, p.285-294, 1980). Anexample of a promoter that acts site-specifically is the promoter ofpetunia chalcone synthase (CHS) gene which works strongly only in thepetals (Plant Molecular Biology, vol.15, p.95-109, 1990). Theabove-mentioned DNA fragment can be expressed in plants by ligating sucha promoter. When a DNA which encodes the polypeptide having said enzymeactivity is cloned from the genomic DNA, it may have been linked with aninherent promoter, and in such cases, there is no need to further linkit with another promoter.

[0031] Further, efficient expression can be expected by introducing aterminator for the termination of transcription at the site downstreamof the region encoding the polypeptide having said enzyme activity (EMBOJournal, vol.7, p.791-799, 1988).

[0032] In order to select plant cells or plants in which the DNA hasbeen introduced, it is preferable to introduce an appropriate markerinto the DNA. Examples of such markers include the kanamycin resistancegene and the hygromycin resistance gene (Plant Molecular Biology, vol.5,p.299-302, 1985). When a microorganism belonging to the genusAgrobacterium is used to introduce the DNA into plant cells or plants,it is necessary to attach the border sequences derived from Ti plasmidat both ends of the sequence to be inserted into plant chromosomes(Nature, vol.313, p.191-196, 1985). Further, it is necessary to link theinsert sequence with a sequence that allows stable retention of plasmidsin a cell of a microorganism belonging to the genus Agrobacterium. Anexample of an expression vector for plants which meets theabove-mentioned requirements is pBI121 (Clonetech Co., Ltd.).

[0033] Examples of methods for introducing said DNA fragment inserted ina vector as described above into plants and obtaining genetically stabletransformed plants include: 1) a method for dicotyledons in which theDNA is introduced via Agrobacterium tumefaciens, the bacterium causingcrown gall disease (Methods in Enzymology, vol.118, p.627-640, 1986); 2)a method in which the DNA is pelted in conjunction with microparticlesof substances such as gold and tungsten at plant cells at a high speedto be incorporated into cell nuclei and then into chromosomes (thehigh-speed microparticle method; Plant Molecular Biology, vol.11,p.433-439, 1989; Bio/Technology, vol.9, p.1080-1085, 1991); and 3) amethod in which the DNA is introduced in conjunction with calciumchloride and polyethylene glycol into protoplasts which have beenprepared with cell wall-degrading enzymes (Nature, vol.296, p.72-74,1982; Nature, vol.319, p.791-793, 1986). The method 1) can beefficiently carried out by incorporating the insert DNA into a binaryvector such as pBI121 (Nucleic Acids Research, vol.12, p.8711-8721,1984). According to the method 2), the DNA can be introduced into plantswhich cannot be infected with a microorganism belonging to the genusAgrobacterium such as monocotyledons. After the introduction of said DNAfragment incorporated into a vector into plant cells according to themethods described above, plant cells in which the introduced DNA isstably retained in the chromosome are selected by utilizing appropriatemarker genes such as those for drug resistance. By inducing thedifferentiation of such plant cells, transformed plants having novelpigment patterns can be obtained.

[0034] In the thus obtained transformed plants, the DNA fragmentsintroduced are retained with genetic stability. In other words, said DNAfragments can be maintained semi-persistently through propagation byvegetative reproduction, or by seeds obtained through self-pollinationor cross pollination.

[0035] Further, it is possible to breed new cultivars which have pigmentpatterns different from those of the first-generation transformants bycrossing the transformants with conventional cultivars to combine theirgenes.

[0036] Thus, a technique is provided which enables the production ofunprecedented cultivars having blue or purple flowers by allowing plantshaving no anthocyanin pigments whose B ring is hydroxylated at both the3′- and 5′-positions, for example, roses and carnations, to synthesizesuch pigments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037]FIG. 1 shows the core sequence which is common to more than 80% ofthe known amino acid sequences for cytochrome P450.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1

[0038] PCR Amplification and Isolation of the Core Sequences ofCytochrome P450 Genes and Their Sequencing

[0039] (1) Synthesis of Primers

[0040] A part of the gene sequence of cytochrome P450 was amplified andisolated by the polymerase chain reaction (PCR) in the following manner.Cytochrome P450 forms a super family consisting of various molecularspecies, but the similarity in the amino acid sequence among thesemolecular species is not so high. However, the sequences of theheme-binding region (core sequence) are relatively common.

[0041] More than 80% of the sequences for cytochrome P450 ever isolatedhave the core sequence shown in FIG. 1. DNA sequences which could encodethe amino acid sequence of the region indicated by arrows werehypothesized. Then, in order to amplify and isolate the DNA sequencesencoding this region by the PCR method, primer DNAs were chemicallysynthesized using the DNA synthesizer, Cyclone Plus™ (manufactured byMilligen/Biosearch). Thus, 16 types of sense primers shown by SEQ ID NO:2 to 17, and 12 types of antisense primers shown by SEQ ID NO: 18 to 29were synthesized.

[0042] The sense primers are synthetic DNA primers each consisting of 18bases, and each primer has, at the 3′ end side, one of the 16 types of8-base DNA sequences at the 3′ end which encode Pro-Phe-Gly orPro-Phe-Ser, and has, at the 5′ end side, a 10-base DNA sequence whichincludes a recognition site for a restriction enzyme, EcoRI. Theantisense primers are synthetic DNA primers each consisting of 18 bases,and each primer has, at the 3′ end side, one of the 12 types ofsequences which are inversely linked to the 8-base DNA sequence at the3′ end encoding Cys-Xxx-Gly (wherein Xxx represents Ile, Leu, Val, Ala,or Pro), and has, at the 5′ end side, a 10-base DNA sequence including arecognition site for a restriction enzyme, BamHI.

[0043] Each primer was used in a 5 μM aqueous solution.

[0044] (2) Extraction of mRNAs from the Petals of Petunia

[0045] Extraction of mRNAs from the petals of petunia was carried outaccording to a modification of the method described in AnalyticalBiochemistry, vol.163, p.16-20, 1987. That is, petals were cut off frombuds of petunia [Petunia hybrida cv. Falcon Blue (Sakata SeedCorporation)] which had been grown in a greenhouse. Ten grams (wetweight) of the petals was put into a mortar, frozen by pouring liquidnitrogen, and then ground with a pestle. To the ground petals were added20 ml of RNA extraction buffer [8 M guanidine hydrochloride, 20 mM Mesbuffer (pH 7.0), 20 mM EDTA, 50 mM mercaptoethanol] and then 10 ml ofphenol/chloroform/isoamyl alcohol (25:24:1) mixture, and mixed well. Theresulting mixture was centrifuged at 10,000×g for 10 minutes, and theupper layer was collected and mixed well with 20 ml ofphenol/chloroform/isoamyl alcohol (25:24:1) mixture. The resultingmixture was centrifuged at 10,000×g for 10 minutes, and the upper layerwas collected. Then, 14 ml of ethanol and 4 ml of 1 M acetic acid wereadded to the upper layer, and the mixture was allowed to stand at −70°C. for one hour, followed by centrifugation at 10,000×g for 10 minutes.The precipitate was separated, dissolved in 10 ml of water, and thenmixed with 3 ml of 10 M lithium chloride. The resulting mixture wasallowed to stand at 4° C. for 2 hours, and centrifuged at 10,000×g for10 minutes. The precipitate was separated, washed with 10 ml of 70%ethanol, and then dried under vacuum. The dried product was dissolved in1 ml of elution buffer [10 mM Tris hydrochloride buffer (pH 7.5), 1 mMEDTA, 0.1% sodium dodecyl sulfate (SDS)], and then subjected topurification using 200 μl of oligotex™-dT 30 (Takara Shuzo Co., Ltd.)according to the instructions provided by the manufacturer to give about3 μg of poly(A)mRNA.

[0046] (3) Synthesis of cDNA from Petunia Petal mRNA

[0047] A cDNA was synthesized from oligo dT primer using theabove-mentioned mRNA as the template and the cDNA Synthesis System PlusRPN1256 (Amersham Co., Ltd.) according to the instructions provided bythe manufacturer. About 2 μg of double strand cDNA was obtained.

[0048] (4) PCR Amplification of the Consensus Sequence of CytochromeP450

[0049] The above-mentioned cDNA (1 ng) as a template DNA was dissolvedin 25 μl of PCR buffer [10 mM Tris hydrochloride buffer (pH 8.3), 1.5 mMmagnesium chloride, 25 mM potassium chloride, 0.05% Tween 20, 100 μMdATP, 100 μM dCTP, 100 μM dGTP, 100 μM dTTP]. The solution was put in a0.5-ml microcentrifugation tube, and as primers, 1 μl of a sense primer(one type) and 1 μl of an antisense primer (one type) both of which wereprepared in the step (1) were added thereto. To the mixture was added0.5 unit of Taq DNA polymerase (Perkin-Elmer Cetus), and 10 μl ofmineral oil was layered over the mixture. The reaction was carried outusing the DNA Thermal Cycler (Perkin-Elmer Cetus) with the cycle programset as follows; 30 seconds at 93° C. and 1 minute at 37° C. for 3cycles, followed by 30 seconds at 93° C. and 1 minute at 55° C. for 37cycles.

[0050] PCR was carried out under the above conditions for each of allthe 192 combinations of 16 sense primers and 12 antisense primers.

[0051] PCR was carried out by reference to PCR Technology, edited by H.A. Ehrlich, Stockton Press, 1989, and PCR Protocols, edited by M. A.Innis, D. H. Gerfand, J. J. Sninsky, and T. J. White, Academic Press,1990.

[0052] (5) Cloning of PCR Products

[0053] The products of the above reaction were subjected to 10%polyacrylamide gel electrophoresis and stained with ethidium bromideaccording to the method described in the book by Maniatis et al. As aresult, a DNA band of approximately 50 bp was detected for 23 among the192 combinations of sense primers and antisense primers. Portionscontaining the DNA band were cut out from the gel, and DNAs wereextracted and purified according to the methods described in the book byManiatis et al. Each of the obtained DNAs was dissolved in 50 μl of Hbuffer [50 mM Tris hydrochloride buffer (pH 7.5), 10 mM magnesiumchloride, 1 mM dithiothreitol, 100 mM sodium chloride]. To the solutionwere added 10 units of the restriction enzyme BamHI (Takara Shuzo Co.,Ltd.) and 10 units of the restriction enzyme EcoRI (Takara Shuzo Co.,Ltd.), and the reaction was carried out at 37° C. for 3 hours. Afteraddition of 150 μl of ethanol, the reaction mixture was allowed to standat −80° C. for 10 minutes, and then centrifuged at 10,000×g for 10minutes. The obtained precipitate was washed with 200 μl of 70% ethanoland dried under vacuum. The obtained DNA was dissolved in 10 μl of TEbuffer [10 mM Tris hydrochloride buffer (pH 7.5), 1 mM EDTA].

[0054] The plasmid vector pUC19 (Takara Shuzo Co., Ltd.) (5 μg) wasdissolved in 50 μl of H buffer, and 10 units of the restriction enzymeBamHI (Takara Shuzo Co., Ltd.) and 10 units of the restriction enzymeEcoRI (Takara Shuzo Co., Ltd.) were added. The reaction was carried outat 37° C. for 3 hours. After addition of 150 μl of ethanol, the reactionmixture was allowed to stand at −80° C. for 10 minutes, and thencentrifuged at 10,000×g for 10 minutes. The obtained precipitate waswashed with 200 μl of 70% ethanol and dried under vacuum. The obtainedvector DNA was dissolved in 100 μl of TE buffer.

[0055] The vector solution thus prepared (1 μl) was mixed with thesolution containing the DNA fragment of approximately 50 bp (10 μl)prepared above, and subjected to ligation at 16° C. for 30 minutes usingthe DNA Ligation Kit (Takara Shuzo Co., Ltd.) according to theinstructions provided by the manufacturer. The volume of the reactionmixture was 60 μl. Highly competent cells of E. coli JM109 (Toyobo Co.,Ltd.) were transformed with 2 μl of the reaction mixture according tothe instructions provided by the manufacturer. According to the methoddescribed in the book by Maniatis et al., the cells were cultured at 37°C. for 20 hours on X-gal ampicillin LB agar medium [1% Bacto Tryptone(Difco Laboratories), 0.5% Bacto Yeast Extract (Difco Laboratories), 1%sodium chloride, 40 μg/ml X-gal, 40 μg/mlisopropyl-1-thio-β-D-galactopyranoside (IPTG), 100 μg/ml ampicillin,1.5% Bacto Agar (Difco Laboratories)]. One of the formed white colonieswas isolated and cultured, and plasmid DNA was extracted from theculture and purified.

[0056] (6) Determination of DNA Sequences of PCR Products

[0057] The nucleotide sequence of the insert fragment in each of the 108clones prepared as described above were determined using the TaqDideoxy™ Terminator Cycle Sequencing Kit (ABI) and the Model 373A DNASequencing System (ABI) according to the instructions provided by themanufacturer. As a result, 15 types of core sequences shown by SEQ IDNO: 30 to 44 were determined for the cytochrome P450 genes.

EXAMPLE 2

[0058] Production of Petunia Backcrossed Population

[0059] (1) Analysis of Pigments in the Petals

[0060] Pigments in the petals were analyzed after convertinganthocyanins into anthocyanidins according to the method described inPhytochemical Methods, Second Edition, edited by J. B. Harbone, p.64,Chapman and Hall, 1989. That is, 0.1 to 0.5 g of the petals was cut offand 1 ml of 2N hydrochloric acid was added. The mixture was heated at95° C. for 40 minutes, and then brought to room temperature. Afteraddition of 300 μl of ethyl acetate followed by thorough mixing, themixture was allowed to stand still, and the upper ethyl acetate layerwas discarded. The residue was heated at 80° C. for 3 minutes toevaporate ethyl acetate, and then brought to room temperature. Afteraddition of 100 μl of isoamyl alcohol followed by thorough mixing, themixture was allowed to stand still, and the upper isoamyl alcohol layerwas collected. Aliquots of 1 to 5 μl of the obtained solution werespotted on a cellulose thin layer plate (Merck & Co., Inc.) andchromatographed using Solvent 1 (conc.hydrochloric acid:aceticacid:water=3:30:10) or Solvent 2 (n-butanol:acetic acid:water=4:1:5) toidentify anthocyanidins based on the Rf values and coloration of thepigment spots. Separately, analysis was also carried out using theHitachi Ion Chromato System (Model L6200 pump and Model L4200 detector),YMC-Pack ODS-A Reversed Phase Column (YMC), and mobile phase consistingof water, acetic acid and methanol (71:10:19) [New High PerformanceLiquid Chromatography, Application II, p.528, Hirokawa Shoten, 1983].Anthocyanidins were identified by using commercially available cyanidin,delphinidin, peonidin, and maruvidin (all produced by Extrasynthese) asstandards.

[0061] (2) Production of Petunia Backcrossed Population

[0062] Crossing of petunia was carried out according to the methoddescribed in Petunia, edited by K. C. Sink, p.180-202, Springer Verlag,1984. A blue flower petunia cultivar, Purple Joy (NPI Seeds) was crossedwith a red flower petunia cultivar, Falcon Red (Sakata Seed Corporation)to obtain hybrids. The hybrids were backcrossed with Falcon Red, andanthocyanidins in the petals of the obtained hybrids were analyzed. Ahybrid plant which had delphinidin as the anthocyanidin component wasselected and then backcrossed with Falcon Red. After such backcrossingwas repeated four times in total, anthocyanidins in the petals of 18plants of the obtained hybrid population were analyzed. Among them tenhybrids had delphinidin (delphinidin-type) as anthocyanidin and eighthybrids had cyanidin (cyanidin-type). The color of petals of the formertype was grayish purple, and that of the latter was red.

[0063] (3) Detection of Flavonoid-3′,5′-hydroxylase Activity

[0064] Detection of flavonoid-31,5′-hydroxylase activity was carried outaccording to a modification of the method described in Z. Naturforsch,vol.37c, p.19-23, 1982. That is, 5 g (wet weight) of petals of buds wasdisrupted using mortar and pestle at 0° C., with 2.5 g of quartz sand(Sigma), 2.5 g of Dow X 1×2 (The Dow Chemical), and 10 ml of buffer forenzyme extraction [0.1 M potassium phosphate buffer (pH 7.5), 20%glycerol, 10 mg/ml sodium ascorbate]. After centrifugation at 12,000×gfor 20 minutes, the obtained supernatant (10 ml) was mixed with 0.4 mlof 1 M magnesium chloride. The mixture was allowed to stand at 0° C. for10 minutes, and centrifuged at 17,000×g for 20 minutes to obtainprecipitate. The precipitate was suspended in a small quantity of thebuffer for enzyme extraction to make a final volume of 500 μl, and thesuspension was used as the microsome fraction.

[0065] An aliquot of 100 μl of the microsome fraction was mixed with 400μl of a reaction mixture [0.1 M potassium phosphate buffer (pH 7.5), 20%glycerol, 10 mg/ml sodium ascorbate, 0.25 mM NADPH (Sigma), 0.25 mMdihydroquercetin (Sigma)], and allowed to react at 25° C. for 30minutes. After addition of 250 μl of ethyl acetate, the mixture wasallowed to stand still, and the upper layer (ethyl acetate layer) wascollected, followed by evaporation of ethyl acetate. The residue wasdissolved in 10 μl of ethyl acetate, and an aliquot of 5 μl of thesolution was spotted on a cellulose thin layer plate (Merck & Co., Inc.)and chromatographed using Solvent 3 (chloroform:aceticacid:water=10:9:1). Flavonoids detected under the UV light wereidentified based on the Rf values. As a result, it was shown thatdihydroquercetin had been converted into dihydromyricetin by the actionof flavonoid-3′,5′-hydroxylase.

[0066] Among the plants of the above-mentioned population obtained bybackcrossing, said enzyme activity was detected in the delphinidin-typeplants. On the other hand, it was not detected in the cyanidin-typeplants. Further, said enzyme activity was detected in Falcon Blue(Sakata Seed Corporation) and Purple Joy (NPI Seeds), which were blueflower petunia cultivars, but was not detected in Falcon Red (SakataSeed Corporation) and Falcon Salmon (Sakata Seed Corporation), whichwere red flower petunia cultivars.

EXAMPLE 3

[0067] SSP.PCR Using the Core Sequence of Cytochrome P450

[0068] (1) Synthesis of K Primers

[0069] On the basis of 15 types of the core sequences for cytochromeP450 shown by SEQ ID NO: 30 to 44 which were obtained in Example 1 (6),15 types of PCR primers shown by SEQ ID NO: 45 to 59 were chemicallysynthesized using the DNA Synthesizer Cyclone Plus (Milligen/Biosearch).Each primer was used in a 5 μM aqueous solution. The primers were namedK01 to K15 primers, respectively, and collectively referred to as Kprimers. K primers are synthetic DNA primers each having a 17-basesequence which starts from the codon for glycine located at theC-terminus of the amino acid sequence of the core and extends toward theN-terminus, and correspond to the sequences from the 32nd nucleotide tothe 16th nucleotide in the core DNA sequences shown by SEQ ID NO: 30 to44.

[0070] (2) Synthesis of a Cassette and a Primer for the Cassette

[0071] Oligonucleotides indicated by SEQ ID NO: 60 and 61 werechemically synthesized using the DNA Synthesizer Cyclone Plus(Milligen/Biosearch), and a 20 μM aqueous solution of eacholigonucleotide was prepared. After 100 μl each of the solutions weremixed, the mixture was heated at 95° C. for 10 minutes, and then kept at50° C. for one hour to obtain a double strand DNA, which is called acassette. One end of the cassette forms a cohesive end of CG-protrudingtype, and therefore, can be efficiently linked with a restriction enddigested with restriction enzymes, such as HinPI, MaeII, MspI andTthHB8I.

[0072] Separately, a 5 μM aqueous solution of the oligonucleotide shownby SEQ ID NO: 60 was prepared and used as the primer for the cassette.

[0073] (3) Synthesis of Petunia Petal cDNA

[0074] Four plants of the delphinidin-type and two plants of thecyanidin-type were selected from the backcrossed population produced inExample 2, and cDNAs were synthesized using mRNAs extracted from thepetals of each plant according to the methods described in Example 1 (2)and (3). Similarly, cDNAs were synthesized from petals of Falcon Blue,Falcon Red, Falcon Salmon, and Purple Joy.

[0075] (4) TthHB8I Digestion of cDNA and Linkage to Cassette

[0076] An aliquot of 0.1 μg of each of the ten types of cDNAs obtainedin (3) above was dissolved in 50 μl of H buffer, and one unit of therestriction enzyme TthHB8I (Takara Shuzo Co., Ltd.) was added. Thereaction was carried out at 65° C. for one hour. Then, the reactionmixture was mixed with 5 μl of phenol/chloroform (1:1) mixture, followedby addition of 150 ml of ethanol. The resulting mixture was allowed tostand at −80° C. for 10 minutes, and centrifuged at 10,000×g for 10minutes. The obtained precipitate was washed with 200 μl of 70% ethanol,and dried under vacuum. The obtained DNA was dissolved in 9 μl of TEbuffer.

[0077] After adding 1 μl of the cassette to each DNA solution, ligationreaction was carried out at 16° C. for 30 minutes using the DNA LigationKit (Takara Shuzo Co., Ltd.) according to the instructions provided bythe manufacturer. The volume of the reaction mixture was 60 μl.

[0078] (5) PCR Between K Primers and the Primer for Cassette

[0079] By the use of each of the above reaction mixtures as a template,the sequence to a near restriction site can be amplified by PCR betweena K primer (01 to 15) and the primer for the cassette.

[0080] An aliquot of 1 μl of each of the above-mentioned reactionmixtures, which was used as a template, was added to 1 μl of a K primerand 1 μl of the primer for the cassette, and mixed with 25 μl of PCRbuffer. The mixture was transferred into a 0.5-ml microcentrifugationtube, 0.5 unit of Taq DNA polymerase (Perkin-Elmer Cetus) was addedthereto, and 10 μl of mineral oil was layered over the mixture. Thereaction was carried out using the DNA Thermal Cycler (Perkin-ElmerCetus) for 40 cycles with the cycle profile consisting of 30 seconds at93° C. and 1 minute at 55° C. According to the methods described in thebook by Maniatis et al., the PCR products were subjected to 10%polyacrylamide gel electrophoresis, and DNA bands were stained withethidium bromide and examined under UV light.

[0081] As a result, in the SSP.PCR using K14 primer, a DNA band of about85 bp was obtained when one of the six types of cDNAs obtained fromFalcon Blue, Purple Joy, and four delphinidin-type backcrossed plantswas used as the template. On the other hand, the band was not detectedwhen one of the four types of cDNAs obtained from Falcon Red, FalconSalmon, and two cyanidin-type backcrossed plants was used as thetemplate. That is, it was demonstrated that the presence/absence of theSSP.PCR products of about 85 bp was genetically linked to thepresence/absence of said enzyme activity. When the other primers wereused, no such product was detected. As the K14 primer was designed basedon the core sequence shown by SEQ ID NO: 43, it was suggested that thesequence shown by SEQ ID NO: 43 was a part of the DNA sequence encodingthe polypeptide which had said enzyme activity.

[0082] (6) Synthesis of J14 Primer

[0083] On the basis of the core sequence of cytochrome P450 shown by SEQID NO: 43, according to which K14 primer was synthesized, a primer shownby SEQ ID NO: 62 was chemically synthesized using the DNA SynthesizerCyclone Plus (Milligen/Biosearch). The primer was named J14 primer, andused in a 5 μM aqueous solution.

[0084] (7) HinPI Digestion of cDNA and Linkage to Cassette

[0085] An aliquot of 0.1 μg of each of the ten types of cDNAs obtainedin (3) above was dissolved in 50 μl of M buffer [10 mM Trishydrochloride buffer (pH 7.5), 10 mM magnesium chloride, 1 mMdithiothreitol, 50 mM sodium chloride], and one unit of the restrictionenzyme HinPI (New England Biolabs) was added. The reaction was carriedout at 37° C. for one hour. Then, the reaction mixture was mixed with 5μl of phenol/chloroform (1:1) mixture, followed by addition of 150 μl ofethanol. The resulting mixture was allowed to stand at −80° C. for 10minutes, and centrifuged at 10,000×g for 10 minutes. The obtainedprecipitate was washed with 200 μl of 70% ethanol, and dried undervacuum. The obtained DNA was dissolved in 9 μl of TE buffer. Afteradding 1 μl of the cassette to each DNA solution, ligation reaction wascarried out at 16° C. for 30 minutes using the DNA Ligation Kit (TakaraShuzo Co., Ltd.) according to the instructions provided by themanufacturer. The volume of the reaction mixture was 60 μl.

[0086] (8) PCR Between J14 Primer and the Primer for Cassette

[0087] An aliquot of 1 μl of each of the above-mentioned reactionmixtures, which was used as a template, was added to 1 μl of J14 primerand 1 μl of the primer for the cassette, and mixed with 25 μl of PCRbuffer. The mixture was transferred into a 0.5-ml microcentrifugationtube, 0.5 unit of Taq DNA polymerase (Perkin-Elmer Cetus) was addedthereto, and 10 μl of mineral oil was layered over the mixture. Thereaction was carried out using the DNA Thermal Cycler (Perkin-ElmerCetus) for 40 cycles with the cycle profile consisting of 30 seconds at93° C. and 1 minute at 55° C. According to the methods described in thebook by Maniatis et al., the PCR products were subjected to 10%polyacrylamide gel electrophoresis, and DNA bands were stained withethidium bromide and examined under UV light.

[0088] As a result, in the SSP.PCR using J14 primer, a DNA band of about280 bp was obtained when one of the six types of cDNAs obtained fromFalcon Blue, Purple Joy, and four delphinidin-type backcrossed plantswas used as the template. On the other hand, the band was not detectedwhen one of the four types of cDNAs obtained from Falcon Red, FalconSalmon, and two cyanidin-type backcrossed plants was used as thetemplate. That is, it was demonstrated that the presence/absence of theSSP.PCR products of about 280 bp was genetically linked to thepresence/absence of said enzyme activity. It was strongly suggested thatthe core sequence shown by SEQ ID NO: 43 was a part of the DNA sequenceencoding the polypeptide which had said enzyme activity.

EXAMPLE 4

[0089] Construction and Sequencing of Plasmid pEAK14

[0090] A library is constructed by incorporating petunia petal cDNAsinto an appropriate vector. The library is searched using the SSP.PCRproduct of about 280 bp obtained in Example 3 as a probe, and thesequence of a clone which hybridizes with the probe is determined.

[0091] (1) Construction of Petunia Petal cDNA Library

[0092] One microgram of cDNA prepared from the petals of petunia (FalconBlue) in Example 1 (3) was cloned using the cDNA Cloning Systemλgt10.RPN1257 (Amersham Co., Ltd.) according to the instructionsprovided by the manufacturer. The final product was subjected to thepackaging reaction using the XDNA in vitro packaging kit Giga Pack Gold(Stratagene Co., Ltd.) according to the instructions provided by themanufacturer. Cells of E. coli NM 514 (Amersham Co., Ltd.) were infectedwith the appropriately diluted packaging products according to theinstructions provided by the manufacturer, and spread on LB agar medium[1% Bacto Tryptone (Difco Laboratories), 0.5% Bacto Yeast Extract (DifcoLaboratories), 1% sodium chloride, 1.5% Bacto Agar (Difco Laboratories)]in plastic plates of 15 cm in diameter (Iwaki Glass Co., Ltd.) to obtainabout 10,000 plaques per plate. A total of five plates were prepared.

[0093] (2) Radiolabeling of DNA Probe

[0094] A portion containing the PCR product of about 280 bp obtained inExample 3 was cut out from the polyacrylamide gel, and the PCR productwas extracted and purified according to the method described in the bookby Maniatis et al. Approximately 50 ng of the purified DNA was labeledwith [α-³²P]dCTP (Amersham Co., Ltd.) using the Multiprime™ DNA LabelingSystem (Amersham Co., Ltd.) according to the instructions provided bythe manufacturer.

[0095] (3) Screening by Plaque Hybridization

[0096] The plaques on the five plates obtained in Example 4 (1) weretransferred onto nylon filters (MSI Co., Ltd.), alkali-denatured, andfixed by heating at 90° C. for 3 hours, according to the methodsdescribed in the book by Maniatis et al. The labeled DNA probe preparedin Example 4 (2) was added to the filters and hybridization was carriedout according to the method described in the book by Maniatis et al. Atthe final step, the filters were washed with 0.1×SSC (15 mM sodiumchloride, 1.5 mM sodium citrate, pH 7.0) at 60° C., and were analyzed byautoradiography to search for positive clones. As a result, 11 positiveclones were obtained. One of the clones was selected, and according tothe methods described in the book by Maniatis et al., phages weremultiplied and DNA was extracted from them.

[0097] (4) Subcloning into Plasmid Vectors

[0098] About 5 μg of the above-mentioned phage DNA was dissolved in 20μl of H buffer, and 10 units of the restriction enzyme BamHI (TakaraShuzo Co., Ltd.) was added. The reaction was carried out at 30° C. for 2hours. After separation of the reaction products by electrophoresisthrough a 0.8% GTG agarose gel (Takara Shuzo Co., Ltd.), a portioncontaining the inserted DNA fragment of about 1.9 kb was cut out. TheDNA fragment was extracted and purified using the SUPREC™-01 (TakaraShuzo Co., Ltd.) according to the instructions provided by themanufacturer.

[0099] The obtained DNA fragment was dissolved in 10 μl of TE buffer and0.2 μg of pUC18 BamHI BAP (Pharmacia Co., Ltd.) was added. Ligationreaction was carried out at 16° C. for 30 minutes using the DNA LigationKit (Takara Shuzo Co., Ltd.) according to the instructions provided bythe manufacturer. The volume of the reaction mixture was 60 μl. Highlycompetent cells of E. coli JM109 (Toyobo Co., Ltd.) were transformedwith 2 μl of the reaction mixture according to the instructions providedby the manufacturer. The cells were cultured at 37° C. for 20 hours onX-gal ampicillin LB agar medium according to the method described in thebook by Maniatis et al. One of the formed white colonies was isolatedand cultured, and plasmid DNA was extracted from the culture andpurified. The obtained plasmid was named pEAK14.

[0100] (5) Determination of DNA Sequence

[0101] The nucleotide sequence of about 1.9 kb which was contained inthe plasmid pEAK14 and derived from petunia cDNA was determined by theModel 373A DNA Sequencing System (ABI Co., Ltd.) using the Deletion Kitfor Kilosequence (Takara Shuzo Co., Ltd.) and the Taq Dideoxy™Terminator Cycle Sequencing Kit (ABI Co., Ltd.) according to theinstructions provided by the manufacturers. The sequence was analyzedusing a sequence analysis software, DNASIS™ (Hitachi SoftwareEngineering Co., Ltd.).

[0102] As a result, the DNA sequence of 1824 bp shown by SEQ ID NO: 1was obtained. This sequence contained an open reading frame starting atthe 116th nucleotide and ending at the 1633rd nucleotide, and coding fora polypeptide consisting of 506 amino acid residues. The amino acidsequence of the polypeptide showed approximately 33% homology to that ofcytochrome P450 of avocado which had been reported (Proc. Natl. Acad.Sci. USA, vol.87, p.3904-3908, 1990).

[0103] This open reading frame was named AK14 sequence.

EXAMPLE 5

[0104] Introduction of the AK14 Sequence into Plant Expression Vectors

[0105] (1) Deletion of ATG Sequence in 5′ Non-coding Region

[0106] An aliquot of 2 μg of the plasmid pEAK14 obtained in Example 4(4) was dissolved in 20 μl of H buffer, and 10 units of the restrictionenzyme BamHI (Takara Shuzo Co., Ltd.) was added. The reaction wascarried out at 30° C. for 2 hours. The reaction products were separatedby electrophoresis through a 0.8% GTG agarose gel (Takara Shuzo Co.,Ltd.), and a portion containing the inserted DNA fragment of about 1.9kb was cut out. Then, the DNA fragment was extracted and purified usingthe SUPREC™-01 (Takara Shuzo Co., Ltd.) according to the instructionsprovided by the manufacturer.

[0107] The obtained DNA fragment was dissolved in 50 μl of BAL31 buffer[20 mM Tris hydrochloride buffer (pH 8.0), 600 mM sodium chloride, 12 mMcalcium chloride, 12 mM magnesium chloride, 1 mM EDTA], and one unit ofBAL31 nuclease S (Takara Shuzo Co., Ltd.) was added. The reaction wascarried out at 30° C. for one minute, followed by addition of 5 μl ofphenol:chloroform (1:1) mixture to terminate the reaction. Afteraddition of 150 μl of ethanol, the reaction mixture was allowed to standat −80° C. for 10 minutes, and then centrifuged at 10,000×g for 10minutes. The obtained precipitate was washed with 200 μl of 70% ethanol,and dried under vacuum. The obtained DNA was dissolved in 50 μl of theKlenow buffer [50 mM Tris hydrochloride buffer (pH 7.5), 10 mM magnesiumchloride, 1 mM dithiothreitol, 100 μM dATP, 100 μM dCTP, 100 μM dGTP,100 μM dTTP], and one unit of Klenow fragment (Takara Shuzo Co., Ltd.)was added. The reaction was carried out at 30° C. for 30 minutes. Afteraddition of 150 μl of ethanol, the reaction mixture was allowed to standat −80° C. for 10 minutes, and then centrifuged at 10,000×g for 10minutes. The obtained precipitate was washed with 200 μl of 70% ethanol,and dried under vacuum. The obtained DNA was dissolved in 10 μl of TEbuffer.

[0108] (2) Subcloning into Plasmid Vectors

[0109] An aliquot of 1 μg of pUC19 (Pharmacia Co., Ltd.) was dissolvedin 50 μl of Sma buffer [10 mM Tris hydrochloride buffer (pH 7.5), 10 mMmagnesium chloride, 1 mM dithiothreitol, 20 mM potassium chloride], and10 units of the restriction enzyme SmaI (Takara Shuzo Co., Ltd.) wasadded. The reaction was carried out at 30° C. for 2 hours. Afteraddition of 150 μl of ethanol, the reaction mixture was allowed to standat −80° C. for 10 minutes, and then centrifuged at 10,000×g for 10minutes. The obtained precipitate was washed with 200 μl of 70% ethanol,and dried under vacuum. The obtained DNA was dissolved in 50 μl of CIPbuffer [50 mM Tris hydrochloride buffer (pH 9.0), 1 mM magnesiumchloride, 0.1 mM zinc chloride, 1 mM spermidine], and 0.1 unit of calfintestine alkaline phosphatase (Boehringer Mannheim GmbH) was added. Thereaction was carried out at 37° C. for 30 minutes, and then at 56° C.for 30 minutes, followed by addition of 5 μl of phenol:chloroform (1:1)mixture to terminate the reaction. After addition of 150 μl of ethanol,the reaction mixture was allowed to stand at −80° C. for 10 minutes, andthen centrifuged at 10,000×g for 10 minutes. The obtained precipitatewas washed with 200 μl of 70% ethanol, and dried under vacuum. Theobtained DNA was dissolved in 10 μl of TE buffer [10 mM Trishydrochloride buffer (pH 7.5), 1 mM EDTA] to obtain a vector DNAsolution.

[0110] An aliquot of 1 μl of the above-mentioned vector DNA solution and2 μl of the DNA solution obtained in Example 5 (1) were mixed andsubjected to ligation at 16° C. for 30 minutes using the DNA LigationKit (Takara Shuzo Co., Ltd.) according to the instructions provided bythe manufacturer. The volume of the reaction mixture was 18 μl. Highlycompetent cells of E. coli JM109 (Toyobo Co., Ltd.) were transformedwith 2 μl of the reaction mixture according to the instructions providedby the manufacturer. The cells were cultured at 37° C. for 20 hours onX-gal ampicillin LB agar medium according to the method described in thebook by Maniatis et al. One of the formed white colonies was isolatedand cultured, and plasmid DNA was extracted from the culture andpurified. The obtained plasmid was named pEAK14S.

[0111] The nucleotide sequence of the region bound to the SmaI sitederived from pUC19 vector in pEAK14S was analyzed. As a result, it wasshown that the sequence of pEAK14S lacked the 1st to the 91stnucleotides of the sequence shown by SEQ ID NO: 1. It was also revealedthat the direction of the insertion was such that the BamHI site ofpUC19 vector was linked to the amino terminus of the AK14 sequence.

[0112] (3) Subcloning into a Plant Expression Vector, pBI121

[0113] An aliquot of 1 μg of pEAK14S was dissolved in 50 μl of M buffer,and 10 units of the restriction enzyme SacI (Takara Shuzo Co., Ltd.) and10 units of the restriction enzyme XbaI (Takara Shuzo Co., Ltd.) wereadded. The reaction was carried out at 37° C. for 2 hours. The reactionproducts were separated by electrophoresis through a 0.8% GTG agarosegel (Takara Shuzo Co., Ltd.), and a portion containing the inserted DNAfragment of about 1.9 kb was cut out. Then, the DNA fragment wasextracted and purified using the SUPREC™-01 (Takara Shuzo Co., Ltd.)according to the instructions provided by the manufacturer. The obtainedDNA fragment was dissolved in 10 μl of TE buffer.

[0114] Similarly, an aliquot of 1 μg of a plant expression vector,pBI121 (GUS Gene Fusion System: Clonetech Co., Ltd.) was dissolved in 50μl of M buffer, and 10 units of the restriction enzyme SacI (TakaraShuzo Co., Ltd.) and 10 units of the restriction enzyme XbaI (TakaraShuzo Co., Ltd.) were added. The reaction was carried out at 37° C. for2 hours. The reaction products were separated by electrophoresis througha 0.8% GTG agarose gel (Takara Shuzo Co., Ltd.), and a portioncontaining the vector DNA fragment of about 11 kb was cut out. Then, thevector DNA fragment was extracted and purified using the SUPREC™-01(Takara Shuzo Co., Ltd.) according to the instructions provided by themanufacturer. The obtained vector DNA fragment was dissolved in 10 μl ofTE buffer.

[0115] An aliquot of 1 μl of the above-mentioned TE buffer containingthe AK14 DNA fragment of about 1.9 kb and 1 μl of the above-mentioned TEbuffer containing the vector DNA fragment of about 11 kb were mixed, andsubjected to ligation at 16° C. for 30 minutes using the DNA LigationKit (Takara Shuzo Co., Ltd.) according to the instructions provided bythe manufacturer. The volume of the reaction mixture was 12 μl. Highlycompetent cells of E. coli JM109 (Toyobo Co., Ltd.) were transformedwith 2 μl of the reaction mixture according to the instructions providedby the manufacturer. The cells were cultured at 37° C. for 20 hours onkanamycin LB agar medium [1% Bacto Tryptone (Difco Laboratories), 0.5%Bacto Yeast Extract (Difco Laboratories), 1% sodium chloride, 50 μg/mlkanamycin, 1.5% Bacto Agar (Difco Laboratories)] according to the methoddescribed in the book by Maniatis et al. One of the formed colonies wasisolated and cultured, and plasmid DNA was extracted from the cultureand purified. The obtained plasmid was named pBAK14.

[0116] (4) Introduction of pBAK14 into Agrobacterium tumefaciens LBA4404

[0117] The plasmid pBAK14 was introduced into Agrobacterium tumefaciensLBA4404 by triparental mating using the GUS Gene Fusion System(Clonetech Co., Ltd.) according to the instructions provided by themanufacturer. E. coli JM109 strain which carries pBAK14 and E. coliHB101 strain which carries pRK2013 (Clonetech Co., Ltd.) were cultured,respectively, in 1 ml of kanamycin LB liquid medium [1% Bacto Tryptone(Difco Laboratories), 0.5% Bacto Yeast Extract (Difco Laboratories), 1%sodium chloride, 50 μg/ml kanamycin] with shaking at 37° C. for 12hours. Separately, Agrobacterium tumefaciens LBA4404 which carriespAL4404 (Clonetech Co., Ltd.) was cultured in 1 ml of streptomycin LBliquid medium [1% Bacto Tryptone (Difco Laboratories), 0.5% Bacto YeastExtract (Difco Laboratories), 1% sodium chloride, 300 μg/mlstreptomycin] with shaking at 28° C. for 36 hours. Three types ofcultured cells were individually collected by centrifugation at 5,000×gfor 10 minutes, washed with 1 ml of water, and suspended in small amountof water. The suspensions were mixed together, and the whole of thecombined suspension was spread on LB agar medium and incubated at 28° C.for 20 hours. The obtained cells were applied on LB agar mediumcontaining 50 μg/ml kanamycin and 300 μg/ml streptomycin, and incubatedat 28° C. over 2 nights. One of the formed colonies was isolated toobtain Agrobacterium tumefaciens LBA4404 carrying both pBAK14 andpAL4404.

EXAMPLE 6

[0118] Introduction of the AK14 Sequence into Tobacco and Its Expression

[0119] (1) Introduction into Tobacco Using a Microorganism of the GenusAgrobacterium

[0120]Agrobacterium tumefaciens LBA 4404 strain carrying pBAK14 andpAL4404 which was obtained in Example 5 was cultured in 10 ml of LBliquid medium [1% Bacto Tryptone (Difco Laboratories), 0.5% Bacto YeastExtract (Difco Laboratories), 1% sodium chloride] containing 50 μg/mlkanamycin and 300 μg/ml streptomycin, with shaking at 28° C. for 40hours. The cultured cells were collected by centrifugation at 5,000×gfor 10 minutes, washed with 10 ml of water, and then suspended in anequal amount of water.

[0121] Leaves of tobacco (Nicotiana tabacum cv. petit Havana SR-1)aseptically subcultured at 25° C. were cut into one centimeter squares,soaked in the above-mentioned cell suspension, and wiped with sterilizedfilter paper. The leaves were placed on MS medium containing 1 μg/ml6-benzyladenine, 0.3 μg/ml 1-naphthaleneacetic acid, 3% sucrose, and0.2% Gelrite (Physiol. Plant., vol.15, p.473-497, 1962) (hereinafterreferred to as the solid PD4 medium) with the abaxial side up, andcultured at 25° C. for 2 days under continuous light illumination at2,500 lux. Then, the leaves were transplanted to the solid PD4 mediumcontaining 500 μg/ml Claforan (for injection, Hoechst Japan Co., Ltd.)and 200 μg/ml kanamycin for culturing, and transplanted to the samemedium every 2 weeks afterward. About one month after the start ofculturing, adventitious buds were induced. The buds were cut off andsubcultured on MS medium containing 500 μg/ml Claforan and 50 μg/mlkanamycin to induce rooting. Plants which took roots were transferredinto pots, after checked for their aseptic condition, and cultivated at25° C. in an artificial weather system. Transgenic plants were thusobtained.

[0122] (2) Detection of Enzyme Activity in Leaves of Tobacco which hadbeen Transformed (hereinafter Referred to as the Transgenic Tobacco)

[0123] Microsome fraction was prepared from 20 g of the transgenictobacco leaves obtained as above according to the method described inExample 2 (3), and flavonoid-3′,5′-hydroxylase activity in the fractionwas determined. As a control, microsome fraction prepared fromnon-transgenic tobacco leaves was used. As a result, said enzymeactivity, which catalyzes the conversion of dihydroquercetin todihydromyricetin, was detected only in the microsome fraction of thetransgenic tobacco.

[0124] (3) Change in Pigments in Petals of the Transgenic Tobacco

[0125] Anthocyanidins were prepared from petals of the transgenic andnon-transgenic tobacco plants, respectively, according to the methoddescribed in Example 2 (1), and analyzed. As a result, only cyanidin wasdetected in the non-transgenic tobacco, whereas cyanidin and delphinidinwere detected in almost the same amounts in the transgenic tobacco.

[0126] The flower colors were compared with The Japan Color Standard ForHorticultural Plants (Japan Color Research Institute). The color offlowers of the transgenic tobacco corresponded to Color No. 8904 or8905, and that of the non-transgenic tobacco corresponded to Color No.9503 or 9504. That is, flowers of the transgenic tobacco showed morebluish color.

EXAMPLE 7

[0127] Introduction of the AK14 Sequence into a Petunia Cultivar withPink Glowers and its Expression

[0128] (1) Introduction into Petunia Using a Microorganism of the GenusAgrobacterium

[0129] Kanamycin-resistant transgenic plants were obtained by infectingleaves of aseptically subcultured petunia (Petunia hybrida cv. FalconPinkvein: Sakata Seed Corporation) with Agrobacterium tumefaciensLBA4404 strain which carries pBAK14 and pAL4404 according to a methodsimilar to that used in Example 6.

[0130] (2) Change in Pigments in Petals of the Transgenic Petunia

[0131] Anthocyanidins were prepared from petals of the above-mentionedtransgenic petunia according to the method described in Example 2 (1),and compared with those prepared from the control, non-transgenicpetunia (Falcon Pinkvein). As a result, little malvidin or delphinidinwas detected in the non-transgenic petunia. On the other hand, thetransgenic petunia had both of them as major components. The majorcomponent in the control plants was peonidin.

[0132] The flower colors at the center area of petals were compared withThe Japan Color Standard For Horticultural Plants (Japan Color ResearchInstitute). The color of flowers of the transgenic petunia correspondedto Color No. 9206 or 9207, and that of the non-transgenic petunia(Falcon Pinkvein) corresponded to Color No. 9204 or 9205. That is,flowers of the transgenic petunia showed more bluish color.

EXAMPLE 8

[0133] Introduction of the AK14 Sequence into Rose and its Expression

[0134] (1) Introduction into Rose Using a Microorganism of the GenusAgrobacterium

[0135] Leaves of aseptically subcultured rose (Rosa hybrida cv. deepred) were infected with Agrobacterium tumefaciens LBA4404 straincarrying pBAK14 and pAL4404 according to a method similar to that usedin Example 6 (1). The leaves were placed on MS medium containing 0.01μg/ml 6-benzyladenine, 10 μg/ml 2,4-dichlorophenoxyacetic acid, 3%sucrose, and 0.2% Gelrite (hereinafter referred to as the solid BEmedium), and cultured at 25° C. for 2 days under continuous lightillumination at 2,500 lux. Then, the leaves were transplanted to thesolid BE medium containing 500 μg/ml Claforan, and after 7 days,transplanted to the solid BE medium containing 500 μg/ml Claforan and200 μg/ml kanamycin. Thereafter, the leaves were transplanted to thesame medium every 2 weeks. After about 2 months, approximately 20 g ofkanamycin-resistant callus was obtained.

[0136] (2) Expression of Enzyme Activity in the Rose Callus

[0137] Microsome fraction was prepared from the callus obtained inExample 8 (1) according to the method described in Example 2 (3), andflavonoid-3′,5′-hydroxylase activity in the fraction was determined. Asa control, microsome fraction prepared from untransformed callus of rosewas used. As a result, said enzyme activity, which catalyzes theconversion of dihydroquercetin to dihydromyricetin, was detected only inthe microsome fraction of the transformed callus.

EXAMPLE 9

[0138] Introduction of the AK14 Sequence into Carnation and itsExpression

[0139] (1) Introduction of pBAK14 into Agrobacterium rhizogenesNIAES1724 Strain

[0140] According to a method similar to that described in Example 5 (4),pBAK14 was introduced into Agrobacterium rhizogenes NIAES1724 strain(obtained from National Institute of Agrobiological Resources, theJapanese Ministry of Agriculture, Forestry and Fisheries). In thisexample, JM103 was used as the E. coli strain, and 25 μg/ml nalidixicacid (Sigma Co., Ltd.) was used instead of streptomycin.

[0141] (2) Introduction of the AK14 Sequence into Carnation Using aMicroorganism of the Genus Agrobacterium

[0142] Petals cut off from buds of carnation (Dianthus caryophillus cv.Nora) were infected with Agrobacterium rhizogenes NIAES1724 carryingpBAK14 according to a method similar to that described in Example 6 (1).The infected petals were placed on solid MS medium containing 0.3 μg/ml6-benzyladenine, 0.3 μg/ml naphthaleneacetic acid, 3% sucrose, and 0.2%Gelrite, and cultured at 25° C. for 3 days under continuous lightillumination at 2,500 lux. Then, the petals were transplanted to thesame medium containing 250 μg/ml Claforan, and after 7 days,transplanted to the same medium containing 250 μg/ml Claforan and 300μg/ml kanamycin. Thereafter, the petals were transplanted to the samemedium every 2 weeks. After about 4 months, approximately 10 g ofkanamycin-resistant hairy roots were obtained.

[0143] (3) Expression of Enzyme Activity in Hairy Roots of Carnation

[0144] Microsome fraction was prepared from the hairy roots obtained inExample 8 (1) according to the method described in Example 2 (3), andflavonoid-3′,5′-hydroxylase activity in the fraction was determined. Asa control, microsome fraction prepared from hairy roots infected withAgrobacterium rhizogenes NIAES1724 strain which did not carry pBAK14 wasused. As a result, said enzyme activity, which catalyzes the conversionof dihydroquercetin to dihydromyricetin, was detected only in themicrosome fraction of the transformed hairy roots.

EXAMPLE 10

[0145] Detection of AK14 Homologous Sequences in Genomic DNAs ofHeterogeneous Plants

[0146] (1) Preparation of Plant Genomic DNA

[0147] Ten to twenty grams of green leaves of each of the followingplants was freeze-dried, and their genomic DNAs were extracted accordingto the method described in DNA Cloning A Practical Approach, vol.2,p.103, 1985, IRL Press: petunia (Petunia hybrida cv. Purple Joy: NPISeeds), nicotiana (Nicotiana affinis cv. F1 Domino: Daiichi Seed Co.,Ltd.), Japanese gentian (Gentiana triflora cv. Japonica), sweet pea(Lathyrus odoratus cv. Royal Deep Blue: Daiichi Seed Co., Ltd.), pansy(Viola tricolor, blue cultivar), primrose (Primula polyantha, purplecultivar), Russell prairie gentian (Eustoma russellianum cv. Royal LightPurple: Takii Seed Co., Ltd.), campanula (Campanula medium, light purplecultivar), delphinium (Delphinium hybridum, pale blue cultivar), andhyacinth (Hyacinthus orientalis, purple cultivar).

[0148] (2) Preparation of Genomic DNA Blots

[0149] An aliquot of 5 μg of each of the genomic DNAs obtained inExample 10 (1) was dissolved in 20 μl of H buffer, and 10 units of therestriction enzyme EcoRV (Takara Shuzo Co., Ltd.) was added. Thereaction was carried out at 37° C. for 2 hours. According to the methoddescribed in the book by Maniatis et al, the digested DNA was subjectedto 0.8% agarose gel electrophoresis, alkali-denatured, and neutralized.Then, the DNA was transferred onto nylon filters (MSI Co., Ltd.), andfixed by heating at 90° C. for 3 hours for fixation to prepare genomicDNA blots.

[0150] (3) Radiolabeling of AK14 Sequence Probe

[0151] An aliquot of 1 μg of pEAK14 obtained in Example 4 was dissolvedin 20 μl of H buffer, and 10 units of the restriction enzyme BamHI(Takara Shuzo Co., Ltd.) was added. The reaction was carried out at 37°C. for 2 hours. The reaction products were separated by electrophoresisthrough a 0.8% GTG agarose gel (Takara Shuzo Co., Ltd.), and a portioncontaining the inserted DNA fragment of about 1.9 kb was cut out. Theinserted DNA fragment was extracted and purified using the SUPREC™-01(Takara Shuzo Co., Ltd.) according to the instructions provided by themanufacturer. An aliquot of 50 ng of the DNA fragment containing theAK14 sequence was labeled with [(α³²P]dCTP (Amersham Co., Ltd.) usingthe Multiprime™ DNA Labeling System (Amersham Co., Ltd.) according tothe instructions provided by the manufacturer.

[0152] (4) Hybridization

[0153] The genomic DNA blots obtained in Example 10 (2) were hybridizedwith the labeled probe of (3) according to the method described in thebook by Maniatis et al. At the final step, the filters were washed twicewith 2×SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0) at 50°C. for 30 minutes. The obtained filters were examined by autoradiographyusing X-ray films (New RX: Fuji Photo Film Co., Ltd.). As a result, theDNAs prepared from petunia (Purple Joy), nicotiana, Japanese gentian,Russell prairie gentian, and campanula showed a clear band. The DNAsprepared from sweet pea and primrose showed a band hybridized with theprobe though unclear. That is, the result showed that homologoussequences which hybridize with the AK14 sequence existed in the genomicDNAs of these plants.

EXAMPLE 11

[0154] Detection of AK14 Homologous Sequences in Petal cDNAs ofHeterogeneous Plants

[0155] (1) Preparation of Petal cDNA

[0156] About 10 g of petals was collected from buds of each of thefollowing plants; petunia (Petunia hybrida cv. Purple Joy: NPI SeedsCo., Ltd.), nicotiana (Nicotiana affinis cv. F1 Domino: Daiichi SeedCo., Ltd.), Japanese gentian (Gentiana triflora cv. Japonica), Russellprairie gentian (Eustoma russellianum cv. Royal Light Purple: Takii SeedCo., Ltd.), and campanula (Campanula medium, light purple cultivar).mRNAs were extracted from the petals according to the method describedin Example 1 (2). By using the obtained mRNAs as templates, doublestrand cDNAs were synthesized using the cDNA Synthesis System PlusRPN1256 (Amersham Co., Ltd.) according to the instructions provided bythe manufacturer.

[0157] (2) Preparation of cDNA Blots

[0158] According to the method described in the book by Maniatis et al,about 0.1 μg of each of the above-mentioned cDNAs was subjected to 0.8%agarose gel electrophoresis, alkali-denatured, and neutralized. Then,the cDNA was transferred onto nylon filters (MSI Co., Ltd.), and fixedby heating at 90° C. for 3 hours to prepare cDNA blots.

[0159] (3) Hybridization

[0160] A radiolabeled AK14 sequence probe was prepared according to amethod similar to that used in Example 10 (3), and hybridized with eachof the above-mentioned cDNA blots according to a method similar to thatused in Example 10 (4). At the final step, the filters were washed twicewith 2×SSC at 50° C. for 30 minutes, and then examined byautoradiography. As a result, each plant showed a clear band at thelocation corresponding to about 2 kb. That is, it was demonstrated thatanalogous sequences which hybridize with the AK14 sequence existed inthe petal cDNAs of these plants.

EXAMPLE 12

[0161] Cloning of the AK14 Homologous Sequence from Russell PrairieGentian and Campanula

[0162] (1) Construction of Petal cDNA Library

[0163] About 20 g of petals was collected from buds of Russell prairiegentian (Eustoma russellianum cv. Royal Light Purple: Takii Seed Co.,Ltd.) and campanula (Campanula medium, light purple cultivar), and mRNAswere extracted from them, respectively, according to the methoddescribed in Example 1 (2). By using the obtained mRNAs as templates,double strand cDNAs were synthesized and cloned into λgt22 vectors usingthe Superscript™ Lambda System (BRL Life Technologies Co., Ltd.)according to the instructions provided by the manufacturer.

[0164] Each final product was subjected to the packaging reaction usingthe XDNA in vitro packaging kit Giga Pack Gold (Stratagene Co., Ltd.)according to the instructions provided by the manufacturer. Cells of E.coli Y1090 (r⁻) (BRL Life Technologies Co., Ltd.) were infected with theappropriately diluted packaging products according to the instructionsprovided by the manufacturer, and spread on LB agar medium [1% BactoTryptone (Difco Laboratories), 0.5% Bacto Yeast Extract (DifcoLaboratories), 1% sodium chloride, 1.5% Bacto Agar (Difco Laboratories)]in plastic plates of 15 cm in diameter (Iwaki Glass Co., Ltd.) to obtainabout 10,000 plaques per plate. Five plates were prepared for Russellprairie gentian and campanula, respectively, to obtain cDNA libraries.

[0165] (2) Screening by Plaque Hybridization

[0166] The plaques on the five plates obtained in Example 4 (1) weretransferred onto nylon filters (MSI Co., Ltd.), alkali-denatured, andfixed by heating at 90° C. for 3 hours according to the methodsdescribed in the book by Maniatis et al. The radiolabeled probe DNAprepared by a method similar to that used in Example 11 (3) was added tothe filters and hybridization was carried out according to the methoddescribed in the book by Maniatis et al. At the final step, the filterswere washed with 2×SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH7.0) at 50° C., and were examined by autoradiography to search forpositive clones. As a result, 12 and 7 positive clones were obtainedfrom the library of Russell prairie gentian and that of campanula,respectively. One clone was selected from each library, and according tothe method described in the book by Maniatis et al, phages weremultiplied and DNAs were extracted from them.

[0167] About 5 μg of each phage DNA was dissolved in 20 μl of H buffer,and 10 units of the restriction enzyme NotI (Takara Shuzo Co., Ltd.) and10 units of the restriction enzyme SalI (Takara Shuzo Co., Ltd.) wereadded. The reaction was carried out at 37° C. for 2 hours. The reactionproducts were separated by electrophoresis through a 0.8% GTG agarosegel (Takara Shuzo Co., Ltd.), and a portion containing the inserted DNAfragment of about 2 kb was cut out from each gel. The DNA fragments wereextracted and purified using the SUPREC™-01 (Takara Shuzo Co., Ltd.)according to the instructions provided by the manufacturer, anddissolved in 10 μl of TE buffer, respectively.

[0168] (3) Subcloning into Plasmid Vectors

[0169] About 1 μg of DNA of a plasmid vector, pBluescriptIIKS+(Stratagene Co., Ltd.) was dissolved in 20 μl of H buffer, and 10 unitsof the restriction enzyme NotI (Takara Shuzo Co., Ltd.) and 10 units ofthe restriction enzyme SalI (Takara Shuzo Co., Ltd.) were added. Thereaction was carried out at 37° C. for 2 hours. The reaction productswere separated by electrophoresis through a 0.8% GTG agarose gel (TakaraShuzo Co., Ltd.), and a portion containing the vector DNA fragment ofabout 3 kb was cut out. The DNA fragment was extracted and purifiedusing the SUPREC™-01 (Takara Shuzo Co., Ltd.) according to theinstructions provided by the manufacturer, and dissolved in 10 μl of TEbuffer.

[0170] To 4 μl each of the two types of inserted DNA fragments obtainedin Example 12 (2) was added 1 μl of the above-mentioned vector DNAfragment, respectively, and ligation was carried out at 16° C. for 30minutes using the DNA Ligation Kit (Takara Shuzo Co., Ltd.) according tothe instructions provided by the manufacturer. The volume of eachreaction mixture was 30 μl. Highly competent cells of E. coli JM109(Toyobo Co., Ltd.) were transformed with 2 μl each of the reactionmixtures, respectively, according to the instructions provided by themanufacturer. The transformed cells were cultured at 37° C. for 20 hourson X-gal ampicillin LB agar medium according to the method described inthe book by Maniatis et al. From each culture, one of the white coloniesformed was isolated and cultured, and plasmid DNA was extracted from theculture and purified. The plasmid derived from the library of Russellprairie gentian was named pETg1, and that from the library of campanulawas named pEKa1.

[0171] (4) Determination of DNA Sequence

[0172] The nucleotide sequences of the DNA fragments which were derivedfrom the petal cDNAs and contained in the plasmids pETg1 and pEKa1 weredetermined by the Model 373A DNA Sequencing System (ABI Co., Ltd.) usingthe Deletion Kit for Kilosequence (Takara Shuzo Co., Ltd.) and the TaqDideoxy™ Terminator Cycle Sequencing Kit (ABI Co., Ltd.) according tothe instructions provided by manufacturers. The sequences were analyzedusing a sequence analysis software, DNASIS™ (Hitachi SoftwareEngineering Co., Ltd.).

[0173] As a result, the DNA sequence of 2174 bp shown by SEQ ID NO: 63was obtained from Russell prairie gentian. This sequence contained anopen reading frame starting at the 92nd nucleotide and ending at the1621st nucleotide, and coding for a polypeptide consisting of 510 aminoacid residues. The amino acid sequence of the polypeptide showed 74%homology to that of AK14. This open reading frame was named Tg1sequence.

[0174] The DNA sequence of 1927 bp shown by SEQ ID NO: 64 was obtainedfrom campanula. This sequence contained an open reading frame startingat the 180th nucleotide and ending at the 1748th nucleotide, and codingfor a polypeptide consisting of 523 amino acid residues. The amino acidsequence of the polypeptide showed 66% homology to that of AK14. Thisopen reading frame was named Ka1 sequence.

EXAMPLE 13

[0175] Introduction of Tg1 and Ka1 into Plant Expression Vectors

[0176] (1) Subcloning into Plant Expression Vector pBI121

[0177] An aliquot of 1 μg of pETg1 was dissolved in 50 μl of H buffer,and 10 units of the restriction enzyme SalI (Takara Shuzo Co., Ltd.) wasadded. The reaction was carried out at 37° C. for 2 hours. Afteraddition of 150 μl of ethanol, the reaction mixture was allowed to standat −80° C. for 10 minutes, and then centrifuged at 10,000×g for 10minutes. The obtained precipitate was washed with 200 μl of 70% ethanol,and dried under vacuum. The obtained DNA was dissolved in 50 μl ofKlenow buffer, and one unit of Klenow fragment (Takara Shuzo Co., Ltd.)was added. The reaction was carried out at 30° C. for 30 minutes. Afteraddition of 150 μl of ethanol, the reaction mixture was allowed to standat −80° C. for 10 minutes, and then centrifuged at 10,000×g for 10minutes. The obtained precipitate was washed with 200 μl of 70% ethanol,and dried under vacuum. The obtained DNA was dissolved in 50 μl of Mbuffer, and 10 units of the restriction enzyme SacI (Takara Shuzo Co.,Ltd.) was added. The reaction was carried out at 37° C. for 2 hours. Thereaction products were separated by electrophoresis through a 0.8% GTGagarose gel (Takara Shuzo Co., Ltd.), and a portion containing theinserted DNA fragment of about 2.2 kb was cut out. The DNA fragment wasextracted and purified using the SUPREC™-01 (Takara Shuzo Co., Ltd.)according to the instructions provided by the manufacturer, anddissolved in 10 μl of TE buffer.

[0178] Separately, 1 μg of pEKa1 was dissolved in 50 μl of H buffer, and10 units of the restriction enzyme SalI (Takara Shuzo Co., Ltd.) wasadded. The reaction was carried out at 37° C. for 2 hours. Afteraddition of 150 μl of ethanol, the reaction mixture was allowed to standat −80° C. for 10 minutes, and then centrifuged at 10,000×g for 10minutes. The obtained precipitate was washed with 200 μl of 70% ethanol,and dried under vacuum. The obtained DNA was dissolved in 50 μl ofKlenow buffer, and one unit of Klenow fragment (Takara Shuzo Co., Ltd.)was added. The reaction was carried out at 30° C. for 30 minutes. Afteraddition of 150 μl of ethanol, the reaction mixture was allowed to standat −80° C. for 10 minutes, and then centrifuged at 10,000×g for 10minutes. The obtained precipitate was washed with 200 μl of 70% ethanol,and dried under vacuum. The obtained DNA was dissolved in 50 μl of Mbuffer, and 0.5 unit of the restriction enzyme SacI (Takara Shuzo Co.,Ltd.) was added. The reaction was carried out at 37° C. for one hour.The reaction products were separated by electrophoresis through a 0.8%GTG agarose gel (Takara Shuzo Co., Ltd.), and a portion containing theinserted DNA fragment of about 1.9 kb was cut out. The DNA fragment wasextracted and purified using the SUPREC™-01 (Takara Shuzo Co., Ltd.)according to the instructions provided by the manufacturer, anddissolved in 10 μl of TE buffer.

[0179] An aliquot of 1 μg of the plant expression vector pBI121 (GUSGene Fusion System: Clonetech Co., Ltd.) was dissolved in 50 μl of Smabuffer [10 mM Tris hydrochloride buffer (pH 7.5), 10 mM magnesiumchloride, 1 mM dithiothreitol, 20 mM potassium chloride], and 10 unitsof the restriction enzyme SmaI (Takara Shuzo Co., Ltd.) was added. Thereaction was carried out at 30° C. for 2 hours. After addition of 150 μlof ethanol, the reaction mixture was allowed to stand at −80° C. for 10minutes, and then centrifuged at 10,000×g for 10 minutes. The obtainedprecipitate was washed with 200 μl of 70% ethanol, and dried undervacuum. The obtained DNA was dissolved in 50 μl of M buffer, and 10units of the restriction enzyme SacI (Takara Shuzo Co., Ltd.) was added.The reaction was carried out at 37° C. for 2 hours. The reactionproducts were separated by electrophoresis through a 0.8% GTG agarosegel (Takara Shuzo Co., Ltd.), and a portion containing the vector DNAfragment of about 11 kb was cut out. The vector DNA fragment wasextracted and purified using the SUPREC™-01 (Takara Shuzo Co., Ltd.)according to the instructions provided by the manufacturer, anddissolved in 10 μl of TE buffer.

[0180] An aliquot of 1 μl of the TE buffer containing the vector DNAfragment and 1 μl of the TE buffer containing the DNA insert fragment ofpETg1 were mixed, and ligation was carried out at 16° C. for 30 minutesusing the DNA Ligation Kit (Takara Shuzo Co., Ltd.) according to theinstructions provided by the manufacturer. The volume of the reactionmixture was 12 μl. Highly competent cells of E. coli JM109 (Toyobo Co.,Ltd.) were transformed with 2 μl of the reaction mixture according tothe instructions provided by the manufacturer. The transformed cellswere cultured at 37° C. for 20 hours on kanamycin LB agar medium [1%Bacto Tryptone (Difco Laboratories), 0.5% Bacto Yeast Extract (DifcoLaboratories), 1% sodium chloride, 50 μg/ml kanamycin, 1.5% Bacto Agar(Difco Laboratories)] according to the method described in the book byManiatis et al. One of the formed colonies was isolated and cultured,and plasmid DNA was extracted and purified. The obtained plasmid wasnamed pBTg1. pBTg1 is a plasmid composed of the plant expression vectorpBI121, and inserted therein, Tg1, which is the AK14 homologous cDNAsequence derived form Russell prairie gentian.

[0181] An aliquot of 1 μl of the TE buffer containing the vector DNAfragment and 1 μl of the TE buffer containing the inserted DNA fragmentof pEKa1 were mixed, and ligation was carried out at 16° C. for 30minutes using the DNA Ligation Kit (Takara Shuzo Co., Ltd.) according tothe instructions provided by the manufacturer. The volume of thereaction mixture was 12 μl. Highly competent cells of E. coli JM109(Toyobo Co., Ltd.) were transformed with 2 μl of the reaction mixtureaccording to the instructions provided by the manufacturer. Thetransformed cells were cultured at 37° C. for 20 hours on kanamycin LBagar medium [1% Bacto Tryptone (Difco Laboratories), 0.5% Bacto YeastExtract (Difco Laboratories), 1% sodium chloride, 50 μg/ml kanamycin,1.5% Bacto Agar (Difco Laboratories)] according to the method describedin the book by Maniatis et al. One of the formed colonies was isolatedand cultured, and plasmid DNA was extracted and purified. The obtainedplasmid was named pBKa1. pBKa1 is a plasmid composed of the plantexpression vector pBI121, and inserted therein, Ka1, which is the AK14homologous cDNA sequence derived from campanula.

[0182] (2) Introduction of pBTg1 and pBKa1 into Agrobacteriumtumefaciens LBA4404 Strain

[0183] The plasmids pBTg1 and pBKa1 were respectively introduced intoAgrobacterium tumefaciens LBA4404 strain using the triparental matingtechnique described in Example 5 (4).

EXAMPLE 14

[0184] Introduction of Tg1 and Ka1 into Tobacco and Their Expression

[0185] (1) Introduction into Tobacco Using a Microorganism of the GenusAgrobacterium

[0186] Leaves of tobacco (Nicotiana tabacum cv. petit Havana SR-1) wereinfected with each of the two types of Agrobacterium strains prepared inExample 13 (2) according to a method similar to that described inExample 6 (1) to obtain kanamycin-resistant transgenic tobacco.

[0187] (2) Detection of Enzyme Activity in Leaves of Transgenic Tobacco

[0188] Microsome fractions were prepared from 20 g each of the leaves oftwo types of transgenic tobacco obtained as above according to themethod described in Example 2 (3), and flavonoid-3′,5′-hydroxylaseactivity in the fractions was determined. As a result, said enzymeactivity, which catalyzes the conversion of dihydroquercetin todihydromyricetin, was detected in the microsome fractions of bothtransgenic tobacco. On the other hand, said enzyme activity was notdetected in the microsome fraction prepared from leaves of thenon-transgenic tobacco.

[0189] (3) Change in Pigments in Petals of the Transgenic Tobacco

[0190] Anthocyanidins were prepared from petals of the transgenic andnon-transgenic tobacco plants, respectively, according to the methoddescribed in Example 2 (1), and analyzed. As a result, only cyanidin wasdetected in the non-transgenic tobacco, whereas cyanidin and delphinidinwere detected in almost the same amounts in both the transgenic tobaccoplants.

[0191] The flower colors were compared with The Japan Color Standard ForHorticultural Plants (Japan Color Research Institute). The color offlowers of the transgenic tobacco corresponded to Color No. 8904 or8905, and that of the non-transgenic tobacco corresponded to Color No.9503 or 9504. That is, flowers of the transgenic tobacco showed morebluish color.

[0192] Industrial Applicability

[0193] According to the present invention, a plant having a pigmentpattern which flowers or fruits of the plant do not originally have canbe provided.

1 67 1824 base pairs nucleic acid double linear cDNA to mRNA Petuniahybrida Falcon Blue CDS 116 to 1633 by experiment 1 GCTACTTCGTTATATATATG TAAAATTGTG ACTTTGAAAA TCATTTAAAT TATCATAAGG60 TTCATTTTATCTTGATCAAA ATATTTACTT CGGCCATATA CGTTTTCCTT TAGTC ATG118 Met 1 ATG CTACTT ACT GAG CTT GGT GCA GCA ACT TCA ATC TTT CTA ATA GCA 166 Met Leu LeuThr Glu Leu Gly Ala Ala Thr Ser Ile Phe Leu Ile Ala 5 10 15 CAC ATA ATCATT TCA ACT CTT ATT TCA AAA ACT ACC GGC CGG CAT CTA 214 His Ile Ile IleSer Thr Leu Ile Ser Lys Thr Thr Gly Arg His Leu 20 25 30 CCG CCG GGG CCAAGA GGG TGG CCG GTG ATC GGA GCA CTT CCA CTT TTA 262 Pro Pro Gly Pro ArgGly Trp Pro Val Ile Gly Ala Leu Pro Leu Leu 35 40 45 GGA GCC ATG CCA CATGTT TCC TTA GCT AAA ATG GCA AAA AAA TAT GGA 310 Gly Ala Met Pro His ValSer Leu Ala Lys Met Ala Lys Lys Tyr Gly 50 55 60 65 GCA ATC ATG TAT CTCAAA GTT GGA ACA TGT GGC ATG GCA GTT GCT TCT 358 Ala Ile Met Tyr Leu LysVal Gly Thr Cys Gly Met Ala Val Ala Ser 70 75 80 ACC CCT GAT GCT GCT AAAGCA TTC TTG AAA ACA CTT GAT ATC AAC TTC 406 Thr Pro Asp Ala Ala Lys AlaPhe Leu Lys Thr Leu Asp Ile Asn Phe 85 90 95 TCC AAT CGT CCA CCT AAT GCAGGT GCC ACT CAC TTA GCT TAT AAT GCT 454 Ser Asn Arg Pro Pro Asn Ala GlyAla Thr His Leu Ala Tyr Asn Ala 100 105 110 CAA GAC ATG GTT TTT GCA CATTAT GGA CCA CGA TGG AAG TTG CTA AGG 502 Gln Asp Met Val Phe Ala His TyrGly Pro Arg Trp Lys Leu Leu Arg 115 120 125 AAA TTA AGC AAC TTG CAT ATGCTA GGG GGA AAA GCC TTA GAG AAT TGG 550 Lys Leu Ser Asn Leu His Met LeuGly Gly Lys Ala Leu Glu Asn Trp 130 135 140 145 GCA AAT GTT CGT GCC AATGAG CTA GGG CAC ATG CTA AAA TCA ATG TCC 598 Ala Asn Val Arg Ala Asn GluLeu Gly His Met Leu Lys Ser Met Ser 150 155 160 GAT ATG AGT CGA GAG GGCCAG AGG GTT GTG GTG GCG GAG ATG TTG ACA 646 Asp Met Ser Arg Glu Gly GlnArg Val Val Val Ala Glu Met Leu Thr 165 170 175 TTT GCC ATG GCC AAT ATGATC GGA CAA GTG ATG CTA AGC AAA AGA GTA 694 Phe Ala Met Ala Asn Met IleGly Gln Val Met Leu Ser Lys Arg Val 180 185 190 TTT GTA GAT AAA GGT GTTGAG GTA AAT GAA TTT AAG GAC ATG GTT GTA 742 Phe Val Asp Lys Gly Val GluVal Asn Glu Phe Lys Asp Met Val Val 195 200 205 GAG TTA ATG ACA ATA GCAGGG TAT TTC AAC ATT GGT GAT TTT ATT CCT 790 Glu Leu Met Thr Ile Ala GlyTyr Phe Asn Ile Gly Asp Phe Ile Pro 210 215 220 225 TGT TTA GCT TGG ATGGAT TTA CAA GGG ATA GAA AAA CGA ATG AAA CGT 838 Cys Leu Ala Trp Met AspLeu Gln Gly Ile Glu Lys Arg Met Lys Arg 230 235 240 TTA CAT AAG AAG TTTGAT GCT TTA TTG ACA AAG ATG TTT GAT GAA CAC 886 Leu His Lys Lys Phe AspAla Leu Leu Thr Lys Met Phe Asp Glu His 245 250 255 AAA GCA ACT ACC TATGAA CGT AAG GGG AAA CCA GAT TTT CTT GAT GTT 934 Lys Ala Thr Thr Tyr GluArg Lys Gly Lys Pro Asp Phe Leu Asp Val 260 265 270 GTT ATG GAA AAT GGGGAC AAT TCT GAA GGA GAA AGA CTC AGT ACA ACC 982 Val Met Glu Asn Gly AspAsn Ser Glu Gly Glu Arg Leu Ser Thr Thr 275 280 285 AAC ATC AAA GCA CTTTTG CTG AAT TTG TTC ACA GCT GGT ACG GAC ACT1030 Asn Ile Lys Ala Leu LeuLeu Asn Leu Phe Thr Ala Gly Thr Asp Thr 290 295 300 305 TCT TCT AGT GCAATA GAA TGG GCA CTT GCA GAA ATG ATG AAG AAC CCT1078 Ser Ser Ser Ala IleGlu Trp Ala Leu Ala Glu Met Met Lys Asn Pro 310 315 320 GCC ATT TTG AAAAAA GCA CAA GCA GAA ATG GAT CAA GTC ATT GGA AGA1126 Ala Ile Leu Lys LysAla Gln Ala Glu Met Asp Gln Val Ile Gly Arg 325 330 335 AAT AGG CGT TTACTC GAA TCC GAT ATC CCA AAT CTC CCT TAC CTC CGA1174 Asn Arg Arg Leu LeuGlu Ser Asp Ile Pro Asn Leu Pro Tyr Leu Arg 340 345 350 GCA ATT TGC AAAGAA ACA TTT CGA AAA CAC CCT TCT ACA CCA TTA AAT1222 Ala Ile Cys Lys GluThr Phe Arg Lys His Pro Ser Thr Pro Leu Asn 355 360 365 CTT CCT AGG ATCTCG AAC GAA CCA TGC ATA GTC GAT GGT TAT TAC ATA1270 Leu Pro Arg Ile SerAsn Glu Pro Cys Ile Val Asp Gly Tyr Tyr Ile 370 375 380 385 CCA AAA AACACT AGG CTT AGT GTT AAC ATA TGG GCA ATT GGA AGA GAT1318 Pro Lys Asn ThrArg Leu Ser Val Asn Ile Trp Ala Ile Gly Arg Asp 390 395 400 CCC CAA GTTTGG GAA AAT CCA CTA GAG TTT AAT CCC GAA AGA TTC TTG1366 Pro Gln Val TrpGlu Asn Pro Leu Glu Phe Asn Pro Glu Arg Phe Leu 405 410 415 AGT GGA AGAAAC TCC AAG ATT GAT CCT CGA GGG AAC GAT TTT GAA TTG1414 Ser Gly Arg AsnSer Lys Ile Asp Pro Arg Gly Asn Asp Phe Glu Leu 420 425 430 ATA CCA TTTGGT GCT GGA CGA AGA ATT TGT GCA GGA ACA AGA ATG GGA1462 Ile Pro Phe GlyAla Gly Arg Arg Ile Cys Ala Gly Thr Arg Met Gly 435 440 445 ATT GTA ATGGTG GAA TAT ATA TTA GGA ACT TTG GTT CAT TCA TTT GAT1510 Ile Val Met ValGlu Tyr Ile Leu Gly Thr Leu Val His Ser Phe Asp 450 455 460 465 TGG AAATTA CCA AGT GAA GTT ATT GAG TTG AAT ATG GAA GAA GCT TTT1558 Trp Lys LeuPro Ser Glu Val Ile Glu Leu Asn Met Glu Glu Ala Phe 470 475 480 GGC TTAGCT TTG CAG AAA GCT GTC CCT CTT GAA GCT ATG GTT ACT CCA1606 Gly Leu AlaLeu Gln Lys Ala Val Pro Leu Glu Ala Met Val Thr Pro 485 490 495 AGG TTACAA TTG GAT GTT TAT GTA CCA TAGCTATAGA TGTGTATTGT 1653 Arg Leu Gln LeuAsp Val Tyr Val Pro 500 505 GCTATAATTG CGCATGTTGT TGGTTGTAGC ATGAGATATTAAAAGGAGTA CATGAAGC1713 ATTGCATGAG TTTAACTTGT AGCTCCTTAA TATTTTAGGTATTTTTCAAT TAATAAGT1773 TTGTTGGTTG GGTAAAAAAA AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA A 1824 18 base pairs nucleic acid single linear Other nucleicacid Synthetic DNA 2 TCGAATTCTN CCATTCGG 18 18 base pairs nucleic acidsingle linear Other nucleic acid Synthetic DNA 3 TCGAATTCTN CCATTTGG 1818 base pairs nucleic acid single linear Other nucleic acid SyntheticDNA 4 TCGAATTCTN CCCTTCGG 18 18 base pairs nucleic acid single linearOther nucleic acid Synthetic DNA 5 TCGAATTCTN CCCTTTGG 18 18 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 6 TCGAATTCTNCCGTTCGG 18 18 base pairs nucleic acid single linear Other nucleic acidSynthetic DNA 7 TCGAATTCTN CCGTTTGG 18 18 base pairs nucleic acid singlelinear Other nucleic acid Synthetic DNA 8 TCGAATTCTN CCTTTCGG 18 18 basepairs nucleic acid single linear Other nucleic acid Synthetic DNA 9TCGAATTCTN CCTTTTGG 18 18 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 10 TCGAATTCTN CCATTCTC 18 18 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 11TCGAATTCTN CCATTTTC 18 18 base pairs nucleic acid single linear GenomicDNA 12 TCGAATTCTN CCCTTCTC 18 18 base pairs nucleic acid single linearOther nucleic acid Synthetic DNA 13 TCGAATTCTN CCCTTTTC 18 18 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 14TCGAATTCTN CCGTTCTC 18 18 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 15 TCGAATTCTN CCGTTTTC 18 18 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 16TCGAATTCTN CCTTTCTC 18 18 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 17 TCGAATTCTN CCTTTTTC 18 18 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 18GCGGATCCCN CCNAAACA 18 18 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 19 GCGGATCCCN CCNAAGCA 18 18 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 20GCGGATCCCN CCNACACA 18 18 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 21 GCGGATCCCN CCNACGCA 18 18 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 22GCGGATCCCN CCNAGACA 18 18 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 23 GCGGATCCCN CCNAGGCA 18 18 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 24GCGGATCCCN CCNATACA 18 18 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 25 GCGGATCCCN CCNATGCA 18 18 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 26GCGGATCCTN CCNGGACA 18 18 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 27 GCGGATCCTN CCNGGGCA 18 18 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 28GCGGATCCCN CCNGCACA 18 18 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 29 GCGGATCCCN CCNGCGCA 18 32 base pairsnucleic acid double linear cDNA to mRNA Petunia hybrida Falcon Blueflower limbs in the bud 30 CCN TTT GGT AGT GGA AGG AGG ATT TGC CCN GG 32Pro Phe Gly Ser Gly Arg Arg Ile Cys Pro Gly 1 5 10 32 base pairs nucleicacid double linear cDNA to mRNA Petunia hybrida Falcon Blue flower limbsin the bud 31 CCN TTT GGT GCT GGA AGA CGT ATA TGT CCN GG 32 Pro Phe GlyAla Gly Arg Arg Ile Cys Pro Gly 1 5 10 32 base pairs nucleic acid doublelinear cDNA to mRNA Petunia hybrida Falcon Blue flower limbs in the bud32 CCN TTT GGT GCT GGT CGA AGA ATA TGC CCN GG 32 Pro Phe Gly Ala Gly ArgArg Ile Cys Pro Gly 1 5 10 32 base pairs nucleic acid double linear cDNAto mRNA Petunia hybrida Falcon Blue flower limbs in the bud 33 CCN TTTGGG ACT GGT CGA CGA ATT TGT CCN GG 32 Pro Phe Gly Thr Gly Arg Arg IleCys Pro Gly 1 5 10 32 base pairs nucleic acid double linear cDNA to mRNAPetunia hybrida Falcon Blue flower limbs in the bud 34 CCN TTT GGC TCGGGA AGA CGA TCT TGT CCN GG 32 Pro Phe Gly Ser Gly Arg Arg Ser Cys ProGly 1 5 10 32 base pairs nucleic acid double linear cDNA to mRNA Petuniahybrida Falcon Blue flower limbs in the bud 35 CCN TTT GGT GCT GGT AGAAGA GTG TGT CCN GG 32 Pro Phe Gly Ala Gly Arg Arg Val Cys Pro Gly 1 5 1032 base pairs nucleic acid double linear cDNA to mRNA Petunia hybridaFalcon Blue flower limbs in the bud 36 CCN TTT GGA GTA GGC CTA AGA ATGTGC CCN GG 32 Pro Phe Gly Val Gly Leu Arg Met Cys Pro Gly 1 5 10 32 basepairs nucleic acid double linear cDNA to mRNA Petunia hybrida FalconBlue flower limbs in the bud 37 CCN TTT GGT GGA GGA CCA CGG CGA TGT CCNGG 32 Pro Phe Gly Gly Gly Pro Arg Arg Cys Pro Gly 1 5 10 32 base pairsnucleic acid double linear cDNA to mRNA Petunia hybrida Falcon Blueflower limbs in the bud 38 CCN TTT GGT GTT GGT AGG AGG AGT TGC CCN GG 32Pro Phe Gly Val Gly Arg Arg Ser Cys Pro Gly 1 5 10 32 base pairs nucleicacid double linear cDNA to mRNA Petunia hybrida Falcon Blue flower limbsin thew bud 39 CCN TTC GGA GTC GGC CCC AAA ATG TGC CCN GG 32 Pro Phe GlyVal Gly Pro Lys Met Cys Pro Gly 1 5 10 32 base pairs nucleic acid doublelinear cDNA to mRNA Petunia hybrida Falcon Blue flower limbs in the bud40 CCN TTC GGT GGA GGA CCA AGA AAA TGC GTN GG 32 Pro Phe Gly Gly Gly ProArg Lys Cys Val Gly 1 5 10 32 base pairs nucleic acid double linear cDNAto mRNA Petunia hybrida Falcon Blue flower limbs in the bud 41 CCN TTCGGC TTT GGT CCT CGA AAA TGC GTN GG 32 Pro Phe Gly Phe Gly Pro Arg LysCys Val Gly 1 5 10 32 base pairs nucleic acid double linear cDNA to mRNAPetunia hybrida Falcon Blue flower limbs in the bud 42 CCN TTT GGC AGTGGT TTC TGT TCA TGT CCN GG 32 Pro Phe Gly Ser Gly Phe Cys Ser Cys ProGly 1 5 10 32 base pairs nucleic acid double linear cDNA to mRNA<Unknown> <Unknown> Petunia hybrida Falcon Blue flower limbs in the bud43 CCN TTT GGT GCT GGA CGA AGA ATT TGT GCN GG 32 Pro Phe Gly Ala Gly ArgArg Ile Cys Ala Gly 1 5 10 32 base pairs nucleic acid double linear cDNAto mRNA Petunia hybrida Falcon Blue flower limbs in the bud 44 CCN TTTGGT GGT GGA AGA AGG ATA TGT CCN GG 32 Pro Phe Gly Gly Gly Arg Arg IleCys Pro Gly 1 5 10 17 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 45 CCNGGGCAAA TCCTCCT 17 17 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 46CCNGGACATA TACGTCT 17 17 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 47 CCNGGGCATA TTCTTCG 17 17 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 48CCNGGACAAA TTCGTCG 17 17 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 49 CCNGGACAAG ATCGTCT 17 17 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 50CCNGGACACA CTCTTCT 17 17 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 51 CCNGGGCACA TTCTTAG 17 17 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 52CCNGGACATC GCCGTGG 17 17 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 53 CCNGGGCAAC TCCTCCT 17 17 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 54CCNGGGCACA TTTTGGG 17 17 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 55 CCNACGCATT TTCTTGG 17 17 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 56CCNACACATT TTCGAGG 17 17 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 57 CCNGGACATG AACAGAA 17 17 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 58CCNGCACAAA TTCTTCG 17 17 base pairs nucleic acid single linear Othernucleic acid Synthetic DNA 59 CCNGGACATA TCCTTCT 17 24 base pairsnucleic acid single linear Other nucleic acid Synthetic DNA 60TGATCCGGAA TTCGTGCCAT CAAG 24 26 base pairs nucleic acid single linearOther nucleic acid Synthetic DNA 61 CGCTTGATGG CACGAATTCC GGATCA 26 15base pairs nucleic acid single linear Other nucleic acid Synthetic DNA62 CCNTTTGGTG CTGGA 15 2174 base pairs nucleic acid double linear cDNAto mRNA Eustoma russellianum CDS 92 to 1621 by experiment 63 GAAAACTATCCATTCTTACC AAGATAAGCA CATTTCTCGT TTCTTTCTAA GAAGAGCATT60 AGGCCAATTCTTTAAGCCCG TACTTAACGA T ATG GCT GTT GGA AAT GGC GTT 112 Met Ala Val GlyAsn Gly Val 1 5 TTA CTT CAC ATT GCT GCA TCA TTG ATG CTG TTC TTT CAT GTGCAA AAA 160 Leu Leu His Ile Ala Ala Ser Leu Met Leu Phe Phe His Val GlnLys 10 15 20 CTT GTG CAA TAT CTA TGG ATG AAT TCC AGG CGC CAC CGG CTT CCACCT 208 Leu Val Gln Tyr Leu Trp Met Asn Ser Arg Arg His Arg Leu Pro Pro25 30 35 GGC CCG ATA GGG TGG CCG GTT CTC GGT GCC CTT CGG CTT TTA GGC ACC256 Gly Pro Ile Gly Trp Pro Val Leu Gly Ala Leu Arg Leu Leu Gly Thr 4045 50 55 ATG CCT CAT GTT GCA CTA GCT AAC ATG GCC AAA AAA TAT GGT CCT GTT304 Met Pro His Val Ala Leu Ala Asn Met Ala Lys Lys Tyr Gly Pro Val 6065 70 ATG TAC TTA AAG GTA GGC AGC TGT GGT CTG GCC GTG GCA TCG ACT CCT352 Met Tyr Leu Lys Val Gly Ser Cys Gly Leu Ala Val Ala Ser Thr Pro 7580 85 GAG GCT GCT AAG GCA TTC CTC AAA ACA CTT GAC ATG AAC TTC TCG AAT400 Glu Ala Ala Lys Ala Phe Leu Lys Thr Leu Asp Met Asn Phe Ser Asn 9095 100 CGG CCG CCT AAT GCA GGG GCT ACC CAT TTG GCC TAT AAT GCT CAG GAC448 Arg Pro Pro Asn Ala Gly Ala Thr His Leu Ala Tyr Asn Ala Gln Asp 105110 115 ATG GTG TTT GCA GAC TAT GGT CCC AGA TGG AAG CTG CTA CGT AAA CTC496 Met Val Phe Ala Asp Tyr Gly Pro Arg Trp Lys Leu Leu Arg Lys Leu 120125 130 135 AGC AAC ATA CAC ATT CTT GGT GGC AAG GCC CTG CAG GGC TGG GAAGAA 544 Ser Asn Ile His Ile Leu Gly Gly Lys Ala Leu Gln Gly Trp Glu Glu140 145 150 GTT CGA AAG AAA GAG CTT GGG TAT ATG CTC TAT GCA ATG GCT GAATCA 592 Val Arg Lys Lys Glu Leu Gly Tyr Met Leu Tyr Ala Met Ala Glu Ser155 160 165 GGG CGA CAT GGC CAG CCA GTG GTG GTG TCA GAG ATG CTA ACA TATGCC 640 Gly Arg His Gly Gln Pro Val Val Val Ser Glu Met Leu Thr Tyr Ala170 175 180 ATG GCA AAC ATG TTA GGC CAA GTG ATG CTC AGC AAG CGA GTT TTCGGG 688 Met Ala Asn Met Leu Gly Gln Val Met Leu Ser Lys Arg Val Phe Gly185 190 195 TCT CAA GGA TCA GAA TCC AAT GAG TTC AAA GAT ATG GTG GTT GAGTTG 736 Ser Gln Gly Ser Glu Ser Asn Glu Phe Lys Asp Met Val Val Glu Leu200 205 210 215 ATG ACT GTT GCT GGC TAT TTC AAC ATA GGT GAT TTT ATC CCCTCG ATT 784 Met Thr Val Ala Gly Tyr Phe Asn Ile Gly Asp Phe Ile Pro SerIle 220 225 230 GCA TGG ATG GAT TTG CAG GGG ATT CAG GGC GGA ATG AAA CGGTTG CAT 832 Ala Trp Met Asp Leu Gln Gly Ile Gln Gly Gly Met Lys Arg LeuHis 235 240 245 AAG AAG TTT GAT GCT TTG TTG ACT CGG TTG CTG GAA GAG CACACT GCA 880 Lys Lys Phe Asp Ala Leu Leu Thr Arg Leu Leu Glu Glu His ThrAla 250 255 260 TCG GCT CAT GAG CGT AAA GGC AGC CCT GAT TTC CTT GAT TTTGTC GTT 928 Ser Ala His Glu Arg Lys Gly Ser Pro Asp Phe Leu Asp Phe ValVal 265 270 275 GCA AAT GGC GAC AAT TCT GAA GGC GAA AGG CTT CAG ACA GTCAAT ATC 976 Ala Asn Gly Asp Asn Ser Glu Gly Glu Arg Leu Gln Thr Val AsnIle 280 285 290 295 AAG GCT CTT TTA TTG AAC ATG TTT ACC GCT GGT ACG GATACA TCT TCA1024 Lys Ala Leu Leu Leu Asn Met Phe Thr Ala Gly Thr Asp ThrSer Ser 300 305 310 AGC GTC ATA GAG TGG GCG CTG GCC GAG TTG CTA AAG AATCCA ATC ATC1072 Ser Val Ile Glu Trp Ala Leu Ala Glu Leu Leu Lys Asn ProIle Ile 315 320 325 CTA AGA CGA GCC CAA GAA GAA ATG GAC GGT GTG ATC GGCCGA GAC CGG1120 Leu Arg Arg Ala Gln Glu Glu Met Asp Gly Val Ile Gly ArgAsp Arg 330 335 340 CGG TTT CTT GAG GCA GAC ATA TCA AAG TTG CCA TAT CTCCAA GCC ATC1168 Arg Phe Leu Glu Ala Asp Ile Ser Lys Leu Pro Tyr Leu GlnAla Ile 345 350 355 TGC AAA GAA GCT TTC AGA AAG CAT CCT TCC ACG CCT TTAAAT CTC CCA1216 Cys Lys Glu Ala Phe Arg Lys His Pro Ser Thr Pro Leu AsnLeu Pro 360 365 370 375 CGA ATC GCG TCG CAA GCA TGT GAA GTA AAT GGA CACTAC ATA CCA AAG1264 Arg Ile Ala Ser Gln Ala Cys Glu Val Asn Gly His TyrIle Pro Lys 380 385 390 GGC ACT AGG CTC AGC GTT AAC ATA TGG GCT ATT GGAAGA GAT CCA TCT1312 Gly Thr Arg Leu Ser Val Asn Ile Trp Ala Ile Gly ArgAsp Pro Ser 395 400 405 GTG TGG GAA AAT CCA AAT GAA TTT AAC CCT GAT AGGTTT TTG GAA CGA1360 Val Trp Glu Asn Pro Asn Glu Phe Asn Pro Asp Arg PheLeu Glu Arg 410 415 420 AAG AAT GCC AAG ATC GAT CCA CGA GGA AAT GAT TTTGAG CTG ATC CCA1408 Lys Asn Ala Lys Ile Asp Pro Arg Gly Asn Asp Phe GluLeu Ile Pro 425 430 435 TTT GGA GCT GGA AGA AGA ATT TGC GCT GGA ACA AGATTG GGA ATA CTT1456 Phe Gly Ala Gly Arg Arg Ile Cys Ala Gly Thr Arg LeuGly Ile Leu 440 445 450 455 CTA GTG GAG TAT ATT TTG GGA ACT TTG GTG CATTCT TTT GTT TGG GAA1504 Leu Val Glu Tyr Ile Leu Gly Thr Leu Val His SerPhe Val Trp Glu 460 465 470 TTG CCA TCC TCT GTG ATT GAA CTT AAC ATG GATGAG TCT TTT GGG CTT1552 Leu Pro Ser Ser Val Ile Glu Leu Asn Met Asp GluSer Phe Gly Leu 475 480 485 GCT CTG CAG AAG GCA GTG CCT CTT GCT GCT ATGGTC ACT CCA CGG CTG1600 Ala Leu Gln Lys Ala Val Pro Leu Ala Ala Met ValThr Pro Arg Leu 490 495 500 CCT CTC CAT ATT TAC TCT CCT TGAGATCTGTGTTCTATGGG TCATTGAGAA 1651 Pro Leu His Ile Tyr Ser Pro 505 510ACAACCGCTG TGTGTTTCTA ACACATGAAT ATGGTTGTGT ACATCTGGCT TATTTATA1711TCCCTATAGA CGAGAAGCCT CGAAGGCAAT GGGGTAATGT TGTTGTTGTC GTGAGACA1771TCTTCTATGT TTCTAAGCAG ATGAGATCTA AGTAGATGAC ATATGCTGTC TTCTACTA1831TTGAAATTAG ATATGCCCCA GAATAAACGC ATCAAACTCG TAATTCGATA CAAAAAAT1891TTGTTGTGGT TTTGAATAAA CACTTATAGA TAATTTGAGA TTTAGAATCG GGTATTTT1951TATATTTTCC ACGTTCATAG GAGTTCGTCC ATGTTTCTGA TTTACAAATA TGATTTTT2011TGGACATTTC TAATAATATC AATTTGTATT CCTGTTTTAA GTTTTTTAAT TTCTCAAG2071TTAGTCCTAA TTAGCAAAGG ACCAGAAAAA CTGTCTAGTT ATGAATCGGG GATAGAAC2131GCAGGAGATG CTGGTTACAA TTTCGATTAA AAAAAAAAAA AAA 2174 1927 base pairsnucleic acid double linear cDNA to mRNA Campanula medium CDS 180 to 1748by experiment 64 ACCAAATGAG CTTTGTAATT TGAGATTAAT CATAATTGCA TGCTCAACTAACATTCTGTA60 TTCATATATC CATATGTATT TTGACCTATA GATATTACAT TACACCTTGAGGCCTTTAT120 TATAGAGAGT GTATCTACTT CCCTTAATAT CACCTTTTCA TTCAACAAGTGAAGCCACC179 ATG TCT ATA GAC ATA TCC ACC CTC TTC TAT GAA CTT GTT GCA GCAATT 227 Met Ser Ile Asp Ile Ser Thr Leu Phe Tyr Glu Leu Val Ala Ala Ile1 5 10 15 TCA CTC TAC TTA GCT ACC TAC TCT TTC ATT CGT TTC CTC TTC AAACCC 275 Ser Leu Tyr Leu Ala Thr Tyr Ser Phe Ile Arg Phe Leu Phe Lys Pro20 25 30 TCT CAC CAC CAC CAC CTC CCT CCC GGC CCA ACC GGA TGG CCG ATC ATC323 Ser His His His His Leu Pro Pro Gly Pro Thr Gly Trp Pro Ile Ile 3540 45 GGA GCC CTT CCA CTC TTA GGC ACC ATG CCA CAT GTT TCC TTA GCC GAC371 Gly Ala Leu Pro Leu Leu Gly Thr Met Pro His Val Ser Leu Ala Asp 5055 60 ATG GCC GTT AAA TAC GGT CCT ATA ATG TAC CTA AAA CTT GGT TCA AAG419 Met Ala Val Lys Tyr Gly Pro Ile Met Tyr Leu Lys Leu Gly Ser Lys 6570 75 80 GGC ACC GTC GTG GCC TCA AAT CCA AAA GCC GCC CGA GCC TTC TTG AAA467 Gly Thr Val Val Ala Ser Asn Pro Lys Ala Ala Arg Ala Phe Leu Lys 8590 95 ACC CAT GAT GCC AAT TTT TCT AAC CGT CCG ATT GAT GGG GGC CCT ACC515 Thr His Asp Ala Asn Phe Ser Asn Arg Pro Ile Asp Gly Gly Pro Thr 100105 110 TAC CTC GCG TAT AAT GCA CAA GAC ATG GTT TTT GCA GAA TAT GGC CCA563 Tyr Leu Ala Tyr Asn Ala Gln Asp Met Val Phe Ala Glu Tyr Gly Pro 115120 125 AAA TGG AAG CTT TTG CGA AAG CTA TGT AGC TTG CAC ATG TTA GGC CCG611 Lys Trp Lys Leu Leu Arg Lys Leu Cys Ser Leu His Met Leu Gly Pro 130135 140 AAG GCA CTC GAG GAT TGG GCT CAT GTC AAA GTT TCA GAG GTC GGT CAT659 Lys Ala Leu Glu Asp Trp Ala His Val Lys Val Ser Glu Val Gly His 145150 155 160 ATG CTC AAA GAA ATG TAC GAG CAA TCG AGT AAG TCA GTG CCA GTGCCA 707 Met Leu Lys Glu Met Tyr Glu Gln Ser Ser Lys Ser Val Pro Val Pro165 170 175 GTG GTG GTG CCA GAG ATG TTA ACT TAT GCC ATG GCT AAT ATG ATTGGA 755 Val Val Val Pro Glu Met Leu Thr Tyr Ala Met Ala Asn Met Ile Gly180 185 190 CGA ATC ATA CTC AGC CGA CGC CCT TTT GTT ATC ACG AGC AAA TTAGAC 803 Arg Ile Ile Leu Ser Arg Arg Pro Phe Val Ile Thr Ser Lys Leu Asp195 200 205 TCG TCT GCT TCT GCT TCT GCT TCT GTT AGT GAA TTC CAA TAT ATGGTT 851 Ser Ser Ala Ser Ala Ser Ala Ser Val Ser Glu Phe Gln Tyr Met Val210 215 220 ATG GAG CTC ATG AGG ATG GCA GGG TTG TTC AAT ATT GGT GAT TTCATA 899 Met Glu Leu Met Arg Met Ala Gly Leu Phe Asn Ile Gly Asp Phe Ile225 230 235 240 CCA TAT ATT GCA TGG ATG GAT TTG CAA GGC ATT CAA CGT GATATG AAG 947 Pro Tyr Ile Ala Trp Met Asp Leu Gln Gly Ile Gln Arg Asp MetLys 245 250 255 GTT ATA CAG AAA AAG TTT GAT GTC TTG TTG AAC AAA ATG ATCAAG GAA 995 Val Ile Gln Lys Lys Phe Asp Val Leu Leu Asn Lys Met Ile LysGlu 260 265 270 CAT ACA GAA TCC GCT CAT GAT CGC AAA GAT AAT CCT GAT TTTCTT GAT1043 His Thr Glu Ser Ala His Asp Arg Lys Asp Asn Pro Asp Phe LeuAsp 275 280 285 ATT CTT ATG GCG GCT ACC CAA GAA AAC ACG GAG GGA ATT CAGCTT AAT1091 Ile Leu Met Ala Ala Thr Gln Glu Asn Thr Glu Gly Ile Gln LeuAsn 290 295 300 CTT GTA AAT GTT AAG GCA CTT CTT TTG GAT TTA TTC ACG GCGGGC ACG1139 Leu Val Asn Val Lys Ala Leu Leu Leu Asp Leu Phe Thr Ala GlyThr 305 310 315 320 GAT ACA TCA TCA AGT GTG ATC GAA TGG GCA CTA GCC GAAATG TTG AAC1187 Asp Thr Ser Ser Ser Val Ile Glu Trp Ala Leu Ala Glu MetLeu Asn 325 330 335 CAT CGA CAG ATC CTA AAC CGG GCC CAC GAA GAA ATG GACCAA GTC ATT1235 His Arg Gln Ile Leu Asn Arg Ala His Glu Glu Met Asp GlnVal Ile 340 345 350 GGC AGA AAC AGA AGA CTA GAA CAA TCT GAC ATA CCA AACTTG CCA TAT1283 Gly Arg Asn Arg Arg Leu Glu Gln Ser Asp Ile Pro Asn LeuPro Tyr 355 360 365 TTC CAA GCC ATA TGC AAA GAA ACA TTC CGA AAA CAC CCTTCC ACG CCC1331 Phe Gln Ala Ile Cys Lys Glu Thr Phe Arg Lys His Pro SerThr Pro 370 375 380 TTA AAC CTC CCA AGA ATC TCA ACA GAA GCA TGT GAA GTGGAC GGA TTT1379 Leu Asn Leu Pro Arg Ile Ser Thr Glu Ala Cys Glu Val AspGly Phe 385 390 395 400 CAC ATA CCA AAA AAC ACT AGA CTA ATA GTG AAC ATATGG GCA ATA GGG1427 His Ile Pro Lys Asn Thr Arg Leu Ile Val Asn Ile TrpAla Ile Gly 405 410 415 AGG GAC CCT AAA GTG TGG GAA AAT CCA TTA GAT TTTACT CCG GAA CGT1475 Arg Asp Pro Lys Val Trp Glu Asn Pro Leu Asp Phe ThrPro Glu Arg 420 425 430 TTC TTG AGT GAA AAA CAC GCG AAA ATT GAT CCG CGAGGT AAT CAT TTT1523 Phe Leu Ser Glu Lys His Ala Lys Ile Asp Pro Arg GlyAsn His Phe 435 440 445 GAG TTA ATC CCA TTT GGG GCT GGA CGA AGG ATA TGTGCA GGG GCT AGA1571 Glu Leu Ile Pro Phe Gly Ala Gly Arg Arg Ile Cys AlaGly Ala Arg 450 455 460 ATG GGA GCG GCC TCG GTC GAG TAC ATA TTA GGT ACATTG GTG CAC TCA1619 Met Gly Ala Ala Ser Val Glu Tyr Ile Leu Gly Thr LeuVal His Ser 465 470 475 480 TTT GAT TGG AAA TTG CCT GAT GGA GTT GTG GAAGTT AAT ATG GAA GAG1667 Phe Asp Trp Lys Leu Pro Asp Gly Val Val Glu ValAsn Met Glu Glu 485 490 495 AGC TTT GGG ATC GCA TTG CAA AAA AAA GTG CCTCTT TCT GCT ATT GTT1715 Ser Phe Gly Ile Ala Leu Gln Lys Lys Val Pro LeuSer Ala Ile Val 500 505 510 ACT CCA AGA TTG CCT CCA AGT TCT TAC ACT GTCTAGGCAAATG CTTATATA1768 Thr Pro Arg Leu Pro Pro Ser Ser Tyr Thr Val 515520 TGAATAATTG ATTGAGTTGT TTAGTTGTAT GAAAGATTTG AGAAAATAAA TTATTAGG1828TTGCACCATT ATGTTGAGAT GGTTGTTGTT AGTGTTAAGG AAGTCGATTG TAGTAATA1888AATTTTATTT TTTTCGAAAA AAAAAAAAAA AAAAAAAAA 1927 506 amino acids aminoacid single linear peptide Petunia hybrida Falcon Blue CDS 116 to 1633by experiment 65 Met 1 Met Leu Leu Thr Glu Leu Gly Ala Ala Thr Ser IlePhe Leu Ile Ala 5 10 15 His Ile Ile Ile Ser Thr Leu Ile Ser Lys Thr ThrGly Arg His Leu 20 25 30 Pro Pro Gly Pro Arg Gly Trp Pro Val Ile Gly AlaLeu Pro Leu Leu 35 40 45 Gly Ala Met Pro His Val Ser Leu Ala Lys Met AlaLys Lys Tyr Gly 50 55 60 65 Ala Ile Met Tyr Leu Lys Val Gly Thr Cys GlyMet Ala Val Ala Ser 70 75 80 Thr Pro Asp Ala Ala Lys Ala Phe Leu Lys ThrLeu Asp Ile Asn Phe 85 90 95 Ser Asn Arg Pro Pro Asn Ala Gly Ala Thr HisLeu Ala Tyr Asn Ala 100 105 110 Gln Asp Met Val Phe Ala His Tyr Gly ProArg Trp Lys Leu Leu Arg 115 120 125 Lys Leu Ser Asn Leu His Met Leu GlyGly Lys Ala Leu Glu Asn Trp 130 135 140 145 Ala Asn Val Arg Ala Asn GluLeu Gly His Met Leu Lys Ser Met Ser 150 155 160 Asp Met Ser Arg Glu GlyGln Arg Val Val Val Ala Glu Met Leu Thr 165 170 175 Phe Ala Met Ala AsnMet Ile Gly Gln Val Met Leu Ser Lys Arg Val 180 185 190 Phe Val Asp LysGly Val Glu Val Asn Glu Phe Lys Asp Met Val Val 195 200 205 Glu Leu MetThr Ile Ala Gly Tyr Phe Asn Ile Gly Asp Phe Ile Pro 210 215 220 225 CysLeu Ala Trp Met Asp Leu Gln Gly Ile Glu Lys Arg Met Lys Arg 230 235 240Leu His Lys Lys Phe Asp Ala Leu Leu Thr Lys Met Phe Asp Glu His 245 250255 Lys Ala Thr Thr Tyr Glu Arg Lys Gly Lys Pro Asp Phe Leu Asp Val 260265 270 Val Met Glu Asn Gly Asp Asn Ser Glu Gly Glu Arg Leu Ser Thr Thr275 280 285 Asn Ile Lys Ala Leu Leu Leu Asn Leu Phe Thr Ala Gly Thr AspThr 290 295 300 305 Ser Ser Ser Ala Ile Glu Trp Ala Leu Ala Glu Met MetLys Asn Pro 310 315 320 Ala Ile Leu Lys Lys Ala Gln Ala Glu Met Asp GlnVal Ile Gly Arg 325 330 335 Asn Arg Arg Leu Leu Glu Ser Asp Ile Pro AsnLeu Pro Tyr Leu Arg 340 345 350 Ala Ile Cys Lys Glu Thr Phe Arg Lys HisPro Ser Thr Pro Leu Asn 355 360 365 Leu Pro Arg Ile Ser Asn Glu Pro CysIle Val Asp Gly Tyr Tyr Ile 370 375 380 385 Pro Lys Asn Thr Arg Leu SerVal Asn Ile Trp Ala Ile Gly Arg Asp 390 395 400 Pro Gln Val Trp Glu AsnPro Leu Glu Phe Asn Pro Glu Arg Phe Leu 405 410 415 Ser Gly Arg Asn SerLys Ile Asp Pro Arg Gly Asn Asp Phe Glu Leu 420 425 430 Ile Pro Phe GlyAla Gly Arg Arg Ile Cys Ala Gly Thr Arg Met Gly 435 440 445 Ile Val MetVal Glu Tyr Ile Leu Gly Thr Leu Val His Ser Phe Asp 450 455 460 465 TrpLys Leu Pro Ser Glu Val Ile Glu Leu Asn Met Glu Glu Ala Phe 470 475 480Gly Leu Ala Leu Gln Lys Ala Val Pro Leu Glu Ala Met Val Thr Pro 485 490495 Arg Leu Gln Leu Asp Val Tyr Val Pro 500 505 510 amino acids aminoacid single linear peptide Eustoma russellianum CDS 92 to 1621 byexperiment 66 Met Ala Val Gly Asn Gly Val 1 5 Leu Leu His Ile Ala AlaSer Leu Met Leu Phe Phe His Val Gln Lys 10 15 20 Leu Val Gln Tyr Leu TrpMet Asn Ser Arg Arg His Arg Leu Pro Pro 25 30 35 Gly Pro Ile Gly Trp ProVal Leu Gly Ala Leu Arg Leu Leu Gly Thr 40 45 50 55 Met Pro His Val AlaLeu Ala Asn Met Ala Lys Lys Tyr Gly Pro Val 60 65 70 Met Tyr Leu Lys ValGly Ser Cys Gly Leu Ala Val Ala Ser Thr Pro 75 80 85 Glu Ala Ala Lys AlaPhe Leu Lys Thr Leu Asp Met Asn Phe Ser Asn 90 95 100 Arg Pro Pro AsnAla Gly Ala Thr His Leu Ala Tyr Asn Ala Gln Asp 105 110 115 Met Val PheAla Asp Tyr Gly Pro Arg Trp Lys Leu Leu Arg Lys Leu 120 125 130 135 SerAsn Ile His Ile Leu Gly Gly Lys Ala Leu Gln Gly Trp Glu Glu 140 145 150Val Arg Lys Lys Glu Leu Gly Tyr Met Leu Tyr Ala Met Ala Glu Ser 155 160165 Gly Arg His Gly Gln Pro Val Val Val Ser Glu Met Leu Thr Tyr Ala 170175 180 Met Ala Asn Met Leu Gly Gln Val Met Leu Ser Lys Arg Val Phe Gly185 190 195 Ser Gln Gly Ser Glu Ser Asn Glu Phe Lys Asp Met Val Val GluLeu 200 205 210 215 Met Thr Val Ala Gly Tyr Phe Asn Ile Gly Asp Phe IlePro Ser Ile 220 225 230 Ala Trp Met Asp Leu Gln Gly Ile Gln Gly Gly MetLys Arg Leu His 235 240 245 Lys Lys Phe Asp Ala Leu Leu Thr Arg Leu LeuGlu Glu His Thr Ala 250 255 260 Ser Ala His Glu Arg Lys Gly Ser Pro AspPhe Leu Asp Phe Val Val 265 270 275 Ala Asn Gly Asp Asn Ser Glu Gly GluArg Leu Gln Thr Val Asn Ile 280 285 290 295 Lys Ala Leu Leu Leu Asn MetPhe Thr Ala Gly Thr Asp Thr Ser Ser 300 305 310 Ser Val Ile Glu Trp AlaLeu Ala Glu Leu Leu Lys Asn Pro Ile Ile 315 320 325 Leu Arg Arg Ala GlnGlu Glu Met Asp Gly Val Ile Gly Arg Asp Arg 330 335 340 Arg Phe Leu GluAla Asp Ile Ser Lys Leu Pro Tyr Leu Gln Ala Ile 345 350 355 Cys Lys GluAla Phe Arg Lys His Pro Ser Thr Pro Leu Asn Leu Pro 360 365 370 375 ArgIle Ala Ser Gln Ala Cys Glu Val Asn Gly His Tyr Ile Pro Lys 380 385 390Gly Thr Arg Leu Ser Val Asn Ile Trp Ala Ile Gly Arg Asp Pro Ser 395 400405 Val Trp Glu Asn Pro Asn Glu Phe Asn Pro Asp Arg Phe Leu Glu Arg 410415 420 Lys Asn Ala Lys Ile Asp Pro Arg Gly Asn Asp Phe Glu Leu Ile Pro425 430 435 Phe Gly Ala Gly Arg Arg Ile Cys Ala Gly Thr Arg Leu Gly IleLeu 440 445 450 455 Leu Val Glu Tyr Ile Leu Gly Thr Leu Val His Ser PheVal Trp Glu 460 465 470 Leu Pro Ser Ser Val Ile Glu Leu Asn Met Asp GluSer Phe Gly Leu 475 480 485 Ala Leu Gln Lys Ala Val Pro Leu Ala Ala MetVal Thr Pro Arg Leu 490 495 500 Pro Leu His Ile Tyr Ser Pro 505 510 523amino acids amino acid single linear peptide Campanula medium CDS 180 to1748 by experiment 67 Met Ser Ile Asp Ile Ser Thr Leu Phe Tyr Glu LeuVal Ala Ala Ile 1 5 10 15 Ser Leu Tyr Leu Ala Thr Tyr Ser Phe Ile ArgPhe Leu Phe Lys Pro 20 25 30 Ser His His His His Leu Pro Pro Gly Pro ThrGly Trp Pro Ile Ile 35 40 45 Gly Ala Leu Pro Leu Leu Gly Thr Met Pro HisVal Ser Leu Ala Asp 50 55 60 Met Ala Val Lys Tyr Gly Pro Ile Met Tyr LeuLys Leu Gly Ser Lys 65 70 75 80 Gly Thr Val Val Ala Ser Asn Pro Lys AlaAla Arg Ala Phe Leu Lys 85 90 95 Thr His Asp Ala Asn Phe Ser Asn Arg ProIle Asp Gly Gly Pro Thr 100 105 110 Tyr Leu Ala Tyr Asn Ala Gln Asp MetVal Phe Ala Glu Tyr Gly Pro 115 120 125 Lys Trp Lys Leu Leu Arg Lys LeuCys Ser Leu His Met Leu Gly Pro 130 135 140 Lys Ala Leu Glu Asp Trp AlaHis Val Lys Val Ser Glu Val Gly His 145 150 155 160 Met Leu Lys Glu MetTyr Glu Gln Ser Ser Lys Ser Val Pro Val Pro 165 170 175 Val Val Val ProGlu Met Leu Thr Tyr Ala Met Ala Asn Met Ile Gly 180 185 190 Arg Ile IleLeu Ser Arg Arg Pro Phe Val Ile Thr Ser Lys Leu Asp 195 200 205 Ser SerAla Ser Ala Ser Ala Ser Val Ser Glu Phe Gln Tyr Met Val 210 215 220 MetGlu Leu Met Arg Met Ala Gly Leu Phe Asn Ile Gly Asp Phe Ile 225 230 235240 Pro Tyr Ile Ala Trp Met Asp Leu Gln Gly Ile Gln Arg Asp Met Lys 245250 255 Val Ile Gln Lys Lys Phe Asp Val Leu Leu Asn Lys Met Ile Lys Glu260 265 270 His Thr Glu Ser Ala His Asp Arg Lys Asp Asn Pro Asp Phe LeuAsp 275 280 285 Ile Leu Met Ala Ala Thr Gln Glu Asn Thr Glu Gly Ile GlnLeu Asn 290 295 300 Leu Val Asn Val Lys Ala Leu Leu Leu Asp Leu Phe ThrAla Gly Thr 305 310 315 320 Asp Thr Ser Ser Ser Val Ile Glu Trp Ala LeuAla Glu Met Leu Asn 325 330 335 His Arg Gln Ile Leu Asn Arg Ala His GluGlu Met Asp Gln Val Ile 340 345 350 Gly Arg Asn Arg Arg Leu Glu Gln SerAsp Ile Pro Asn Leu Pro Tyr 355 360 365 Phe Gln Ala Ile Cys Lys Glu ThrPhe Arg Lys His Pro Ser Thr Pro 370 375 380 Leu Asn Leu Pro Arg Ile SerThr Glu Ala Cys Glu Val Asp Gly Phe 385 390 395 400 His Ile Pro Lys AsnThr Arg Leu Ile Val Asn Ile Trp Ala Ile Gly 405 410 415 Arg Asp Pro LysVal Trp Glu Asn Pro Leu Asp Phe Thr Pro Glu Arg 420 425 430 Phe Leu SerGlu Lys His Ala Lys Ile Asp Pro Arg Gly Asn His Phe 435 440 445 Glu LeuIle Pro Phe Gly Ala Gly Arg Arg Ile Cys Ala Gly Ala Arg 450 455 460 MetGly Ala Ala Ser Val Glu Tyr Ile Leu Gly Thr Leu Val His Ser 465 470 475480 Phe Asp Trp Lys Leu Pro Asp Gly Val Val Glu Val Asn Met Glu Glu 485490 495 Ser Phe Gly Ile Ala Leu Gln Lys Lys Val Pro Leu Ser Ala Ile Val500 505 510 Thr Pro Arg Leu Pro Pro Ser Ser Tyr Thr Val 515 520

1) A DNA encoding a polypeptide which has flavonoid-3′,5′-hydroxylaseactivity and which is represented by the amino acid sequence shown bySEQ ID NO: 1, 63 or 64, or a DNA which hybridizes with said DNA. 2) TheDNA according to claim (1), wherein a part of the nucleotide sequence ofsaid DNA is deleted or replaced by another nucleotide sequence. 3) Arecombinant DNA composed of a vector DNA and the DNA of claim (1) orclaim (2) which is inserted in the vector DNA. 4) A plant or plant cellwhich carries the recombinant DNA according to claim (3). 5) The plantor plant cell according to claim (4), wherein said plant belongs to thegenus Rosa, Nicotiana, Petunia, or Dianthus. 6) A DNA which hybridizeswith a DNA represented by the nucleotide sequence shown by SEQ ID NO: 1,63 or 64 in 2×SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0)at 50° C. 7) A method for producing a plant, which comprises:introducing a recombinant DNA composed of a vector DNA fragment and aDNA fragment which encodes a polypeptide havingflavonoid-3′,5′-hydroxylase activity into a plant; breeding a plantwhich can express a pigment based on the genetic information of the DNAencoding said polypeptide; and harvesting said plant thus obtained. 8)The method for producing a plant according to claim (7), wherein saidDNA fragment which encodes the polypeptide havingflavonoid-3′,5′-hydroxylase activity is the DNA of claim (1) or claim(2). 9) A DNA which has the nucleotide sequence shown by any of SEQ IDNO: 2 to
 29. 10) A DNA which has a sequence comprising a sequenceidentical with the eight-nucleotide sequence from the 3′-terminus in thesequence of the DNA of claim (9). 11) A method for amplifying andisolating a gene fragment which encodes the amino acid sequence of theheme-binding region of cytochrome P450 enzyme, by polymerase chainreaction (PCR) using the DNA of claim (9) or claim (10) as primers.